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.     

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.     

Development of B-Genome Chromosome Addition Lines of B. napus Using Different Interspecific Brassica Hybrids

By interspecific hybridization within the genus Brassica, trigenomic haploids were produced and back-crossed four times with B. napus, variety ‘Andor’. From this material, monosomic B-genome chromosome addition lines were selected with the extra chromosome derived from three different B-genome sources, i.e., B. nigra (BB), B. carinata (BBCC), and B. juncea (AABB). After selfing and/or microspore culture, disomic addition lines were obtained. Meiotic behavior was studied of the trigenomic hybrids, the pentaploid BC₁ plants, and the monosomic addition lines. The addition lines were shown to possess cytological stability and good fertility.     

Utility of AFLP markers for the assessment of genetic diversity within Brassica nigra germplasm

Genetic diversity of 18 Brassica nigra accessions was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two B. rapa and two B. juncea accessions were selected as outliers in the study. Eight AFLP primer combinations generated a total of 426 bands, of which 79% were polymorphic. The UPGMA method was employed to construct a dendrogram based on the Jaccard's similarity coefficient. The accessions of B. rapa separated from those of B. nigra at a genetic similarity coefficient of 0.27 while those of B. juncea did so at 0.5. The genetic similarity coefficients within the B. nigra accessions ranged from 0.58 to 0.86. Based on these coefficients it was concluded that the B. nigra accessions show high levels of genetic variation. These results have significant implications in the crop improvement programmes for the agronomically important crop B. juncea, an amphidiploid of B. nigra and B. rapa. Two incorrectly labelled B. nigra accessions were also identified. These accessions were found to cluster with those of B. juncea accessions. This result demonstrates the great value of AFLP markers in the management of genebanks.     

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)     

Mapping of Molecular Markers on Brassica B-Genome Chromosomes Added to Brassica napus

Using interspecific hybridization among various Brassica species, B-genome chromosomes from different sources of Brassica, i.e. B. nigra (BB, 2n = 18), B. carinata (BBCC), 2n = 34) and B. juncea (AABB, 2n = 36) were transferred into the Canadian variety ‘Andor’ of B. napus. Monosomic addition lines were selected (AACC + 1B, 2n = 39) by cytological control. For characterization of the alien chromosomes, series of isozymes, RFLPs and RAPD markers were employed. This permitted the identification of a total of 39 lines representing seven of the eight B-genome chromosomes.     

Inheritance of the resistance to Leptosphaeria maculans of Brassica nigra and B. juncea in near-isogenic lines of B. napus

The inheritance of resistance to Leptosphaeria maculans was studied in near-isogenic lines derived from asymmetric somatic hybrids between Brassica napus+Brassica nigra and Brassica napus+Brassica juncea, respectively. The hybrids had been backcrossed to B. napus for seven generations before the genetic segregation of the blackleg resistance was determined. The results of the inheritance studies suggested that one single dominant allele controls the resistance in the Brassica napojuncea line, whereas two independent dominant loci were found in the Brassica naponigra line. Total leaf DNA from the near-isogenic lines was isolated and 89 loci were detected by hybridization to 66 restriction fragment length polymorphism (RFLP) markers previously mapped in the B. nigra genome. Out of the 89 loci, eight loci were detected in the B. naponigra line and six were found in the B. napojuncea line. RFLP markers co-segregating with blackleg resistance in adult leaves were also found. Two markers associated with linkage group 5 and 8, respectively, of the B genome were found in the B. naponigra line and one marker was associated with linkage group 2 in the B. napojuncea line.     

Assessment of transgene escape from Brassica rapa (B. campestris) into B. nigra or Sinapis arvensis

The possibility of gene transfer between Brassica rapa and the two weedy species B. nigra and Sinapis arvensis was evaluated with the special concern on transgene escape from B. rapa to these two weedy species. B. rapa cultivar Tobin was reciprocally crossed to five and four strains of B. nigra and S. arvensis, respectively, using controlled cross. A single interspecific hybrid was obtained from the cross B. rapa×B. nigra, but no other cross was successful. The fertility of this hybrid on open pollination, selfing and backcrosses was investigated. The data of the present study and the information available to date indicate that gene transfer between B. rapa and B. nigra is possible. The chance of transgene escape from B. rapa to B. nigra depends essentially on whether natural cross can occur between these two species. Gene transfer between B. rapa and S. arvensis is at the most difficult, whereas trans-gene escape directly from B. rapa to S. arvensis appears very unlikely.     

Selection of Diploid Nematode-Resistant Sugar Beet from Monosomic Addition Lines

Monosomic sugar beet/wild beet addition lines (2n = 19) with full resistance against the beet cyst nematode have been characterized in different ways. Within the B. procumbens and B. webbiana addition lines three groups could be classified according to their isozymes pattern, growth habit, transmission rate, and resistance level. It is assumed that B. procumbens and B. webbiana each possess three different chromosomes which carry genes for nematode resistance. In the offspring of the addition lines diploid translocation types appear at very low frequencies, Isozyme pattern or growth type of the resistant plants were used for selecting diploid types in the offspring of monosomic addition lines. Effective selection could be made in progenies of susceptible sugar beets pollinated by addition lines because the pollen transmission of the alien chromosome is very low. Using these methods 7 nematode-resistant sugar beet lines could be selected. The transmission rates of the resistance gene ranged from 70.6% to 100%. Threw heterozygous progenies showed a 1:1 segregation indicating monogenic dominant inheritance of resistance. The level of resistance was as high as in the addition lines.     

Cytoplasmic effects on photosynthesis and ribulose-1,5-bisphosphate carboxylase activity in Brassica species

Brassica genotypes representing four different species viz. B. nigra and B. campestris (both primary diploids), B. juncea and B. carinata (both allotetraploids) were examined for photosynthesis rate (Pn), ribulose-1,5-bisphosphate carboxylase (RuBPC) activity, leaf soluble protein and chlorophyll content. B. nigra is the male and female parent of B. juncea and B. carinata, respectively. B. campestris is the maternal parent of B. juncea. Pn was significantly higher in B. nigra and B. carinata compared to the other two species. These two species also sustained Pn of the leaves for a longer period during ontogeny. When B. nigra was the male parent, the higher Pn of B. nigra was not inherited in B. juncea. On the other hand, the Pn and pattern of ontogenetic changes in B. campestris and B. juncea were more or less similar. In specific RuBPC activity on soluble protein basis, chlorophyll and soluble protein content B. carinata also followed B. nigra whereas, B. juncea followed B. campestris. These results indicated the possible maternal(cytoplasmic) influence on the inheritance of these traits. This revised version was published online in July 2006 with corrections to the Cover Date.     

Utilization of Asymmetric Somatic Hybridization for the Transfer of Disease Resistance from Brassica nigra to Brassica napus

Asymmetric somatic hybrid calli were produced between Brassica napus and a transgenic (Hyg ^R) line of B. nigra using a donor recipient fusion method for the production of cybrids. The transgenic line of B. nigra used as a donor also possessed genetic resistance to the pathogenic fungi Phoma lingam and Plasmodiophora brassicae. Using hygromycin for selection, 332 hybrid calli were obtained from which 30 produced shoots (1—-20 per callus) which were rooted on a hormone-free culture medium. The rooted shoots were transferred into soil and cultivated in a growth chamber where the plants were tested for resistance against the two pathogens. Out of 129 hybrid plants tested for resistance against P. brassicae, 30 (23.3 %) plants proved to be resistant and from 78 plants tested for resistance against P. lingam, 41 (52.6 %) plants remained disease-free after infection.     

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.     

Hybridizations among Brassica napus, B. rapa and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis under open pollination conditions in the field

The rate of natural crosses occurring among the cultivated Brassica species B. napus, B. rapa, and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis was studied in co-cultivation experiments under field conditions in Saskatchewan, Canada, with special reference to evaluation of the possibility of transgene escape from the cultivated to the weedy species. Natural crosses occurred among B. napus, B. rapa, and B. juncea, indicating that hybridizations among these three species do occur under field conditions. On the other hand, no natural crosses occurred between the cultivated species and B. nigra or S. arvensis. It is concluded that the crosses between the cultivated and weedy species are practically impossible under field conditions in Saskatchewan, and that the escape of transgenes from transgenic cultivars of B. napus, B. rapa and B. juncea into B. nigra and S. arvensis is basically zero in this region.     

Electrophoretic Investigations on Nematode Resistant Sugar Beets

Hybridizations were made between beta vulgaris and three wild species of the patellares section being resistant to the best cyst nematode {heterodera schachtii). Monosomic addition lines (2 n = 19) with full nematode resistance were investigated together with wild beets by means of electrophoretical techniques. One alkaline esterase band and a complex of several acidic esterase bands were localized on the resistance-carrying B. procumbens chromosome. The alkaline esterase marker also appeared in B. patellaris addition lines. An aconitase double band was visible in two of four B. webbiana addition lines. One resistant monotelosomic addition line with a small B. procumbens fragment had lost the esterase gene. Evidence is given that more than one chromosome is carrying genes for nematode resistance. The use of electrophoretic screening together with a nematode testing program is discussed.     

Use of a synthetic hexaploid Triticum miguschovae for transfer of leaf rust resistance to common wheat

Summary Triticum miguschovae, a genome addition synthetic, was used as a source for transfer of leaf rust (Puccinia recondita tritici) resistance to common wheat. This synthetic, developed from two wild species Triticum militinae and Aegilops squarrosa, proves a valuable donor of the genes for leaf rust resistance. Leaf rust resistance was transferred from T. miguschovae by both dominant and recessive genes. Stable lines phenotypically similar to their recurrent parents Kavkaz and Bezostaya 1 but differing from them in a high level of leaf rust resistance were obtained. The genes for resistance in 3 selected lines differed from each other and from the known effective genes Lr9, Lr19, and Lr24. The resistance of one of them (line 1229) is controlled by two complementary interacting genes located on chromosome 7B and 1D was revealed by monosomic analysis.     

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.     

Nematode Resistance Derived from Wild Beet and its Meiotic Stability in Sugar Beet

Three different karyotypes of sugar beet with resistance against the beet cyst nematode (Heterodera schachtii) have been investigated. These comprised monosomic addition lines (2n = 19) with one complete chromosome from B. patellarris or B. procumbens, one line with a chromosomal fragment added to the normal sugar beet chromosome complement (2n =18 + fragment) and one diploid line (2n = 18). The fragment originated from a B. procumbens chromosome since during meiosis it formed a univalem. It carries the gene for nematode resistance. Meiotic disturbances like univalems. laggards, anaphase I bridges, fragments and micronuclei were observed in all resistant genotypes. These may result in an exclusion of the chromosome fragment carrying the resistance from the rest of the genome. In the diploid resistant line, a chromosome with a translocation could be distinguished from the other B. vulgaris chromosomes. Meiotic irregularities also appeared in diploid resistant types and are one main reason for low transmission of the resistance. Tin-relationship between meiotic stability and the transmission rate of the resistance gene is discussed.     

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.     

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.