G. Röbbelen

Mapping the genome of rapeseed (Brassica napus L.). I. Construction of an RFLP linkage map and localization of QTLs for seed glucosinolate content

A linkage map of the rapeseed genome comprising 204 RFLP markers, 2 RAPD markers, and 1 phenotypic marker was constructed using a F1 derived doubled haploid population obtained from a cross between the winter rapeseed varieties ‘Mansholts Hamburger Raps’ and ‘Samourai’. The mapped markers were distributed on 19 linkage groups covering 1441 cM. About 43% of these markers proved to be of dominant nature; 36% of the mapped marker loci were duplicated, and conserved linkage arrangements indicated duplicated regions in the rapeseed genome. Deviation from Mendelian segregation ratios was observed for 27.8% of the markers. Most of these markers were clustered in 7 large blocks on 7 linkage groups, indicating an equal number of effective factors responsible for the skewed segregations. Using cDNA probes for the genes of acyl-carrier-protein (ACP) and β-ketoacyl-ACP-synthase I (KASI) we were able to map three and two loci, respectively, for these genes. The linkage map was used to localize QTLs for seed glucosinolate content by interval mapping. Four QTLs could be mapped on four linkage groups, giving a minimum number of factors involved in the genetic control of this trait. The estimated effects of the mapped QTLs explain about 74% of the difference between both parental lines and about 61.7 % of the phenotypic variance observed in the doubled haploid mapping population.

[Contributions to the analysis of the Brassica-genome].

1. Pachytene chromosome structure of the 3 Brassica species B. campestris (genome a; n=10), B. oleracea (genome c; n=9), and B. nigra (genome b; n=8) is described. In each of these species the same 6 chromosome types can be recognized by such structural characteristics as total length, symmetry of arms, and especially shape of the heterochromatic centromere region. 2. B. campestris is doubly tetrasomic for 2 chromosomes of different type and hexasomic for another chromosome type. B. oleracea is a triple tetrasomic, B. nigra a double tretasomic. The chromosome types present in increased numbers are different ones in the 3 species. The formulas for the genomes are: a = AA B C DD E FFF, c = A BB CC D EE F, and b = A B C DD E FF. 3. Secondary paring has been observed in pachytene of a triploid aac-hybrid, proving partial homology in the different genomes of some chromosomes belonging to the same structural type. 4. In each of the 3 genomes the duplicated chromosomes can be recognized by the occurrence of secondary pairing, the frequency of which depends on the degree of structural similarity. Secondary pairing is found in pachytene as well as metaphase I and II. 5. These results agree with earlier observations (see Discussion) and confirm the presence of balanced secondary polyploidy in Brassica derivable from a basic chromosome number x=6.Summary1.Pachytene chromosome structure of the 3 Brassica species B. campestris (genome a; n=10), B. oleracea (genome c; n=9), and B. nigra (genome b; n=8) is described. In each of these species the same 6 chromosome types can be recognized by such structural characteristics as total length, symmetry of arms, and especially shape of the heterochromatic centromere region.2.B. campestris is doubly tetrasomic for 2 chromosomes of different type and hexasomic for another chromosome type. B. oleracea is a triple tetrasomic, B. nigra a double tretasomic. The chromosome types present in increased numbers are different ones in the 3 species. The formulas for the genomes are: a = AA B C DD E FFF, c = A BB CC D EE F, and b = A B C DD E FF.3.Secondary paring has been observed in pachytene of a triploid aac-hybrid, proving partial homology in the different genomes of some chromosomes belonging to the same structural type.4.In each of the 3 genomes the duplicated chromosomes can be recognized by the occurrence of secondary pairing, the frequency of which depends on the degree of structural similarity. Secondary pairing is found in pachytene as well as metaphase I and II.5.These results agree with earlier observations (see Discussion) and confirm the presence of balanced secondary polyploidy in Brassica derivable from a basic chromosome number x=6.

Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores

SummaryThe effect of colchicine on isolated microspore cultures of Brassica napus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chromosome doubling at an early developmental stage, thus avoiding the production of haploid or chimeric plants. Colchicine was added to the culture medium immediately after isolation of B. napus microspores. The cultures were incubated from 6 to 72 h with various concentrations of colchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry.The highest diploidization rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80–90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a lower diploidization rate (76–80%). Treatment durations of 6 h were not long enough to induce a high rate of diploidization, whereas the application of 10 mg/l for 72 h was also very effective.A sample of the plants regenerated from the colchicine treated microspores was transferred to the greenhouse. The plants looked similar to normal diploid rapeseed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessary. The advantage of applying a minimum volume of colchicine under controlled in vitro conditions means a considerable saving of time and labour in DH-breeding programs.

Mapping of a gene determining linolenic acid concentration in rapeseed with DNA-based markers

Rapeseed ranks third in world oil production. An important breeding objective to improve oil quality in this crop is to lower linolenic acid concentration in the seeds. Previous reports indicate that the concentration of this acid in Brassica napus is determined by two or three nuclear genes. Using DNA-based markers, we have successfully mapped a gene determining linolenic acid concentration in an F2 population derived from crossing the cultivar ‘Duplo’ and alow linolenic acid line, 3637-1. Linolenic acid concentration in this population ranged from 2.1% to 10.5% with-amean of 6.2%. A RAPD marker, K01-1100, displayed significantly different frequencies between two subpopulations consisting of either high or low linolenic acid concentration individuals sampled from the two extremes of the F2 distribution. Marker K01-1100 segregated in a codominant fashion when used as an RFLP probe on DNA from individuals of this F2 population. The linolenic acid concentration means for the three resulting RFLP genotypes in the F2 population were 4.8% (homozygous 3637-1 allele), 6.4% (heterozygous), and 7.5% (homozygous ‘Duplo’ allele), respectively. It is estimated that this marker accounts for 26.5% of the genetic variation of linolenic acid concentration in this population.

Inheritance of resistance derived from the B-genome of Brassica against Phoma lingam in rapeseed and the development of molecular markers

Abstract Genes of the B genome of Brassica conditioning Phoma resistance at the epicotyle were transferred into Brassica napus by interspecific hybridization. The recombinant lines expressed high resistance similar to that of the donor parents. Unlike the oligo- or poly-genically inherited resistance of B. napus known so far, the B-genome resistance genes of the recombinant lines behaved monogenically dominant. No significant differences in the level of resistance or in the phenotype of the resistance mechanisms were observed among homozygous resistant plants when the different B-genome origins investigated, i.e. B. nigra, B. juncea and B. carinata, were compared. Therefore it was assumed that the resistance genes of each B-genome species and the resistance mechanisms of the species are identical. Temperature increased the expression of internal lesions caused by Phoma lingam. High summer temperatures in the greenhouse led to faster development of tissue damage at the epicotyle of plants, resulting in significant deviations in segregation ratios, when fixed scores were used for disease classification. Independent of origin, the three B-genome resistance genes were introgressed at the same location of the rapeseed genome. The arrangement and distances of closely linked RFLP markers on linkage-groups were similar to those of the same markers on linkage group six of the rapeseed map. It is concluded that the B-genome resistance genes were introgressed by homoeologous recombination after allosyndetical pairing of B-genome chromosomes with the A- or C-genome chromosomes.

Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers

The three B genomes of Brassica contained in B. nigra, B. carinata and B. juncea were dissected by addition in B. napus. Using phenotypic, isozyme and molecular markers we characterized 8 alien B-genome chromosomes from B. nigra and B. carinata and 7 from B. juncea by constructing synteney groups. The alien chromosomes of the three different sources showed extensive intragenomic recombinations that were detected by the presence of the same loci in more than one synteny group but flanked by different markers. In addition, intergenomic recombinations were observed. These were evident in euploid AACC plants of the rapeseed phenotype derived from the addition lines carrying a few markers from the B genome due to translocations and recombinations between non-homoeologous chromosomes. The high plasticity of the Brassica genomes may have been an powerful factor in directing their evolution by hybridization and amphiploidy.

Increased embryogenesis after colchicine treatment of microspore cultures of Brassica napus L.

Summary Efforts to improve the induction of embryogenesis in microspore culture of B. napus have focussed to date on growth conditions of the donor plants and culture conditions of the microspores. For initiating haploid development, the first pollen mitosis proceeds in a symmetrical fashion in contrast to the asymmetrical division in normal gametogenesis. Earlier studies have shown that disruption of this asymmetrical division by adding colchicine into the culture medium (25 mgL -1 for 12 h) of microspores increased the number of regenerated embryoids. In the present experiments the effect of higher concentrations and longer colchicine treatments have been analyzed in B. napus . Of the 57 microspore culture experiments with four different genotypes, 69% of the experiments produced a positive effect on embryogenesis. The best results were obtained with the treatment of 100 mgL -1 colchicine for 24 h. This produced a 3 fold increase in the average number of regenerated embryoids. The second best result was obtained with the 10 mgL -1 treatment for 3 days, leading to a 1.8 fold increase in regeneration. The individual treatments showed a strong tendency for the 24- h and 72-h duration of treatments to be significantly better than their controls, while the 6-h treatment resulted in a low level of significance. Colchicine concentrations as low as as 10 mgL -1 were sufficient to improve embryogenesis and the results suggest that treatment durations longer than 24h (or even 72h) may positively affect embryogenesis. A negative effect of colchicine on the regeneration rate was observed in 10% of the experiments, indicating that the developmental stage of microspores at the time of colchicine treatment might be important. During the culture of microspores and the regeneration of embryoids no signs of toxicity, e.g. malformations of embryoids, were observed. The experiments confirm that disruption of cytoskeleton components at or before the first pollen mitosis in vitro contributes to an improved embryogenesis in B. napus .

Identification by Giemsa technique of the translocations separating cultivated rye from three wild species of Secale

Complete identification of the translocations involved in evolution of S. vavilovii, S. africanum and S. cereale from S. montanum was attained by meiotic analysis after Giemsa banding technique. Based on the original mitotic karyotype of S. montanum, the different chromosome arms were determined by centromere position and banding pattern of chromosomes for the four species and all of the possible interspecific hybrids. This first consistent scheme of cytogenetic relationships reveals: one translocation each, separating S. montanum from S. vavilovii and S. africanum, two translocations each, separating S. cereale from S. vavilovii and S. africanum, and three translocations each, separating S. cereale from S. montanum and S. africanum, respectively.

13 Breeding: An overview

Publisher Summary This chapter describes the present status and future potential of the plant breeding within the Brassica coenospecies . Plant breeding is a technology based on numerous empirical skills and established practices; however, on critical scientific knowledge, such as that described in the preceding chapters. The aim of plant breeding is the development of plant cultivars with high performance in farm productions and the improvement of those currently grown. The genus Brassica comprises a considerable number of crops of greatly diverging biological characteristics. The extent of this diversity relates to their uses and to the history of their domestication. In the cross pollinated Brassica species, various methods of population improvement are used. In many cases, mass selection has been successful, but this simple method is replaced by advanced types of recurrent selection.

Somatic association at interphase studied by giemsa banding technique

Somatic association of rye chromosomes has been studied by Giemsa banding technique at interphase in wheat-rye addition lines. Telomeres of the rye chromosomes appearing as chromocenters, showed close somatic association in disomic addition lines, but they were distributed at random in double monosomic additions. This demonstrates directly that somatic association of homologues at interphase is even closer in non-dividing nuclei than in metaphase cells, which had been investigated so far. The finding has relevance to the assumption that somatic association phenomena are a prerequisite to the meiotic pairing process.