Development of Yellow Seeded Brassica napus Through Interspecific Crosses

Yellow seeded Brassica napus was developed through interspecific crosses with the two mustard species, B. juncea and B. carinata. The objective of these two interspecific crosses was the introgression of genes for yellow seed colour from the A genome of B. juncea and C genome of B. carinata into the A and C genomes of B. napus, respectively. The interspecific F₁ generations were backcrossed to B. napus in an attempt to eliminate B genome chromosomes and to improve fertility. Backcross F₂ plants of the (B. napus×B. juncea) ×B. napus cross were then crossed with backcross F₂ plants of the (B. napus×B. carinata) ×B. napus cross. The objective of this intercrossing was to combine the A and C genome yellow seeded characteristics of the two backcross populations into one genotype. The F₂ generation of the backcross F₂ intercrosses was grown in the field, plants were individually harvested and visually rated for seed colour. Ninety-one yellow seeded plants were identified among the 4858 plants inspected. This result indicated that the interspecific crossing scheme was successful in developing yellow seeded B. napus.     

Hybridization of Sinapis alba L. and Brassica napus L. via Embryo Rescue

Embryo rescue techniques were used to obtain hybrids between Sinapis alba L. (white mustard) and Brassica napus L. (oilseed rape) with the goal of improving the disease tolerance of oilseed rape. Hybrid plants with 31 or 43 chromosomes were only recovered, when S. alba, was used as the female parent. One hybrid was obtained from the cross S. alba L. cv. ‘Kirby’×B. napus L. cv. ‘Topas’, while 26 hybrids were obtained, when various S. alba L. cultivars were pollinated with the rapid cycling B. napus line CrGC 5006. All F₁, hybrid plants were male sterile; however, the first generation backcross to B. napus L., also obtained by embryo rescue, produced plants with 50 chromosomes and 61–84 % pollen viability. Second backcross generation seed was produced by normal sexual crossing. Preliminary cytological analyses of pollen mother cells of hybrid plants suggests the possibility of genetic exchange between the two species.     

Genomic in situ hybridization analysis of Brassica napus × Orychophragmus violaceus hybrids and production of B. napus aneuploids

To further utilize the valuable germplasm Orychophragmus violaceus for Brassica genetics and breeding, a B. napus × O. violaceus cross was repeated with embryo rescue. All F₁ plants except one B. napus haploid were mixoploids (2n = 17–39 in ovaries) with 2n = 31, 37, 38 and 39 as the maximal chromosome numbers in individuals, but the higher numbers mostly appeared in pollen mother cells (PMCs) with a preponderance of 2n = 30, 37 and 38. Only one chromosome and one chromosome segment of O. violaceus were detected at a low frequency in some ovary cells and PMCs with 2n = 37, 38 and 39 as determined by genomic in situ hybridization analysis. The fatty acid profiles of seeds from the majority of the F₁ and F₂ plants were similar to those of female B. napus cv. ‘Oro’, but some were obviously different in the percentages of oleic, linoleic and erucic acids, and some F₂ plants (2n = 38) with good seed set had high percentages of oleic (>70.0%) or linoleic (to 38.3%) acids and low erucic acid (<1%). Subsequently, many kinds of B. napus aneuploids (2n = 28, 30, 34, 36, 37, 39 and 42), without O. violaceus chromosomes, were derived from F₂ progeny and microspores of partial F₁ plants. Finally, the cytological mechanisms behind the variations in chromosome numbers were discussed together with the implications of these aneuploids for Brassica genome research and of the plants with altered fatty acid profiles for improving the oil quality of B. napus.     

Construction of a molecular functional map of rapeseed (Brassica napus L.) using differentially expressed genes between hybrid and its parents

Brassica napus L. is an important oilseed and fodder crop with significant heterosis for seed yield and other agronomic traits, but very little is known about the molecular basis of heterosis. As an initial step towards understanding the molecular events associated with this phenomenon, a molecular functional map of rapeseed was constructed using differentially expressed genes in hybrid identified by microarrays. Single-strand conformational polymorphism (SSCP) analysis was applied for genetic mapping in an F ₂ population of 184 individuals resulting from crossing ‘`SI-1300 × Eagle’'. A total of 162 markers including 154 loci corresponding to 98 differentially expressed genes assigned to 17 functional categories and 8 SSR markers were grouped into 21 linkage groups (LGs), covering a total map distance of 2267.3 cM. Subsequently, this map was aligned with Arabidopsis thaliana in silico. Comparative mapping shows that genes localized on each Arabidopsis chromosome have orthologs dispreading in different B. napus LGs. Similarly, a majority of LGs were made of homologous genes from different Arabidopsis chromosomes. In addition, a total of 25 syntenic regions were identified in B. napus, in most of which the gene order was not consistent between the two species, and each of the conserved regions in the A. thaliana genome was homologous to 1--5 distinct regions in the B. napus genome. These results indicate that it is not easy to exploit A. thaliana information for B. napus based on synteny.     

Maternal and embryonal control of seed colour by different Brassica alboglabra chromosomes

Most oilseed rape, Brassica napus, cultivars are black‐seeded. The progenitor species, Brassica rapa, has either yellow or black seeds, while known cultivars of the other progenitor species Brassica oleracea/alboglabra have black seeds. To determine which chromosomes of B. alboglabra are carriers of seed colour genes, B. rapaalboglabra monosomic addition lines were produced from a B. napus resynthesized from yellow‐seeded B. rapa and brown/black‐seeded B. alboglabra. Eight out of nine possible lines have been developed and transmission frequencies of the alien chromosomes were estimated. Three B. alboglabra chromosomes in three of these lines influenced seed colour. B. rapa plants carrying alien chromosome 1 exhibited a maternal control of seed colour and produced only brown seeds, which gave rise to plants with either yellow or brown seeds. However, B. rapa plants carrying alien chromosome 4 or another as yet unidentified alien chromosome exhibited an embryonal control of seed colour and produced a mixture of yellow and brown seeds. The yellow seeds gave rise to yellow‐seeded plants, while the brown seeds gave rise to plants that yielded a mixture of yellow and brown seeds, depending on the absence or presence, respectively, of the B. alboglabra chromosome. Consequently, both maternal and embryonal control of seed colour are expected to contribute to the black‐seeded phenotype of oilseed rape.     

Establishing Cytoplasmic Male Sterility in Brassica napus by Mitochondrial Recombination with B. tournefortii

Fusion experiments between B. napus and X-ray-treated B. tournefortii protoplasts were carried out to develop cytoplasmic male sterility (ems) in B. napus. From the regenerants, six lines containing male sterile plants were selected; five lines segregated for male sterility, but one line (25–143) was completely male-sterile from the beginning. Molecular analyses of mitochondrial (mt) and chloroplast (cp) DNA of B. napus, B. tournefortii, B. juncea and cms juncea indicated that the original cytoplasmic donor of the cms juncea-system in B. napus was a B. tournefortii form, while the B. napus genotype used for the fusion experiments had a B. campestris cytoplasm. By analysis (it regenerated plants, line 25–143 was identified as possessing mt-DNA recombined between B. campestris and B. tournefortii. with the major part derived from B. campestris. No differences were detected between epDNAs from H. campestris and from line 25—143. The other five lines were similar to B. campestris with all the probes used. The low frequency of sterile lines from the fusion experiments and the inheritance of the cms in segregating progenies are both discussed.     

Inheritance of seed colour in Brassica campestris L. and breeding for yellow-seeded B. napus L.

Summary Seed colour inheritance was studied in five yellow-seeded and one black-seeded B. campestris accessions. Diallel crosses between the yellow-seeded types indicated that the four var. yellow sarson accessions of Indian origin had the same genotype for seed colour but were different from the Swedish yellow-seeded breeding line. Black seed colour was dominant over yellow. The segregation patterns for seed colour in F₂ (Including reciprocals) and BC₁ (backcross of F₁ to the yellow-seeded parent) indicated that the black seed colour was conditioned by a single dominant gene. Seed colour was mainly controlled by the maternal genotype but influenced by the interplay between the maternal and endosperm and/or embryonic genotypes. For developing yellow-seeded B. napus genotypes, resynthesized B. napus lines containing genes for yellow seed (Chen et al., 1988) were crossed with B. napus of yellow/brown seeds, or with yellow-seeded B. carinata. Yellow-seeded F₂ plants were found in the crosses that involved the B. napus breeding line. However, this yellow-seeded character did not breed true up to F₄. Crosses between a yellow-seeded F₃ plant and a monogenomically controlled black-seeded B. napus line of resynthesized origin revealed that the black-seeded trait in the B. alboglabra genome was possibly governed by two independently dominant genes with duplicated effect. Crossability between the resynthesized B. napus lines as female and B. carinata as male was fairly high. The sterility of the F₁ plants prevented further breeding progress for developing yellow-seeded B. napus by this strategy.     

Successful induction of trigenomic hexaploid <Emphasis Type="Italic">Brassica</Emphasis> from a triploid hybrid of <Emphasis Type="Italic">B.</Emphasis><Emphasis Type="Italic">napus</Emphasis> L. and <Emphasis Type="Italic">B. nigra</Emphasis> (L.) Koch

A triploid hybrid with an ABC genome constitution, produced from an interspecific cross between Brassica napus (AACC genome) and B. nigra (BB genome), was used as source material for chromosome doubling. Two approaches were undertaken for the production of hexaploids: firstly, by self-pollination and open-pollination of the triploid hybrid; and secondly, by application of colchicine to axillary meristems of triploid plants. Sixteen seeds were harvested from triploid plants and two seedlings were confirmed to be hexaploids with 54 chromosomes. Pollen viability increased from 13% in triploids to a maximum of 49% in hexaploids. Petal length increased from 1.3 cm (triploid) to 1.9 cm and 1.8 cm in the two hexaploids and longest stamen length increased from 0.9 cm (triploid) to 1.1 cm in the hexaploids. Pollen grains were longer in hexaploids (43.7 and 46.3 μm) compared to the triploid (25.4 μm). A few aneuploid offsprings were also observed, with chromosome number ranging from 34 to 48. This study shows that trigenomic hexaploids can be produced in Brassica through interspecific hybridisation of B. napus and B. nigra followed by colchicine treatment.     

Intergeneric crosses for the transfer of resistance to the beet cyst nematode from Raphanus sativus to Brassica napus

Summary The possibilities to transfer important traits and in particular resistance to the beet cyst nematode (Heterodera schachtii, abbrev. BCN) from Raphanus sativus to Brassica napus were investigated. For these studies B. napus, R. sativus, the bridging hybrid ×Brassicoraphanus (Raparadish) as well as offspring of the cross ×Brassicoraphanus (Raparadish) ×B. napus were used. Reciprocal crosses between B. napus and R. sativus were unsuccessful, also with the use of embryo rescue. Crosses between ×Brassicoraphanus as female parent and B. napus resulted in a large number of F₁ hybrids, whereas the reciprocal cross yielded mainly matromorphic plants. BC₁, BC₂ and BC₃ plants were obtained from backcrosses with B. napus, which was used as the male parent. F₁ hybrids and BC plants showed a large variation for morphology and male and female fertility. Cuttings of some F₁ and BC₁ plants, obtained from crosses involving resistant plants of ×Brassicoraphanus, were found to possess a level of resistance similar to that of the resistant parent. These results and indications for meiotic pairing between chromosomes of genome R with those of the genomes A and/or C suggest that introgression of the BCN-resistance of Raphanus into B. napus may be achieved.     

An evaluation of the potential of intergeneric gene transfer between Brassica napus and Sinapis arvensis

The possibility of gene transfer between Brassica napus and Sinapis arvensis was evaluated. Six spring-type cultivars of B. napus and four strains of S. arvensis were reciprocally crossed through controlled crosses. No hybrid was yielded from any cross. However, one hybrid with 28 chromosomes was obtained from B. napus×S. arvensis through ovule culture. The hybrid plant was highly sterile and set no seed on open pollination. Two F₂ plants, with 35 and 36 chromosomes respectively, were obtained through self-pollination by hand. Backcross of B. napus produced 23 plants carrying some characteristics of S. arvensis, but backcross to S. arvensis failed to produce a plant. The chromosome counts of the BC₁F₁ plants indicated that gametes with more than nine chromosomes were favoured during the meiosis. The data demonstrated that gene transfer from S. arvensis to B. napus was very difficult under controlled cross and backcross, while to transfer genes from B. napus to S. arvensis would be extremely remote even under the most favorable conditions.     

Development of yellow-seeded Brassica napus of double low quality

Two yellow‐seeded white‐petalled Brassica napus F₇ inbred lines, developed from interspecific crosses, containing 26–28% emcic acid and more than 40 μmol glucosinolates (GLS)/g seed were crossed with two black/dark brown seeded B. napus varieties of double low quality and 287 doubled haploid (DH) lines were produced. The segregation in the DH lines indicated that three to four gene loci are involved in the determination of seed colour, and yellow seeds are formed when all alleles in all loci are in the homozygous recessive state. A dominant gene governed white petal colour and is linked with an erucic acid allele that, in the homozygous condition, produces 26–28% erucic acid. Four gene loci are involved in the control of total GLS content where low GLS was due to the presence of recessive alleles in the homozygous condition in all loci. From the DH breeding population a yellow‐seeded, yellow‐petalled, zero erucic acid line was obtained. This line was further crossed with conventional B. napus varieties of double low quality and, following pedigree selection, a yellow seeded B. napus of double low quality was obtained. The yellow seeds had higher oil plus protein content and lower fibre content than black seeds. A reduction of the concentration of chromogenic substances was found in the transparent seed coat of the yellow‐seeded B. napus.     

Identification of Maintainer Genes in Brassica napus L. for the Male-Sterility-Inducing Cytoplasm of Diplotaxis muralis L.

Male fertility of F¹ interspecific hybrid plants derived from crosses between cytoplasmic male-sterile Brassica campestris in Diplotaxis muralis cytoplasm and 147 B. napus cultivars was Investigated. F¹, plants obtained, from crosses with the B. napus cultivars‘Mangum’and‘Hinchu’were male-sterile while F¹ plants derived from all other crosses were male-fertile. This indicated that these two cultivars carried maintainer genes far the male-sterility-inducing cytoplasm of D. muralis. Sterility was stable In plants derived from backcrosses of male-sterile F; plants with‘Mangun and‘Hinchu’but the seed set of backcross plants was low. With restorer genes readily available in B. napus, these findings could lead to the development of a new cytoplasmic male sterility system for the breeding of B. napus hybrid cultivars.     

A cytogenetic study of the progenies of hybrids between Brassica napus and Brassica oleracea, Brassica bourgeaui, Brassica cretica and Brassica montana

In this cytogenetic study the progeny of all crosses were investigated in F₁, F₂ and backcross (BC₁) hybrids. Brassica napus and F₁ hybrids between B. napus and B. oleracea, and between B. napus and three wild relatives of B. oleracea (B. bourgeaui, B. cretica and B. montana). Each of the wild relatives has 18 somatic chromosomes. Interspecific F₁ hybrids were obtained through ovary culture mean. These had 28 and 37 chromosomes and their mean pollen fertility was 10.7% and 93.0%, respectively. Many F₂ and BC₁ seeds were harvested from the F₁ hybrids with 37 chromosomes after self‐pollination and open pollination of the F₁ hybrids, and backcrossing with B. napus. Many aneuploids were obtained in the F₂ and BC₁ plants. It is evident from these investigations that the F₁ hybrids may serve as bridge plants to improve B. napus and other Brassica crops.     

Introgression of genes into cultivated Brassica napus through resynthesis of B. napus via ovule culture and the accompanying change in fatty acid composition

Fifteen lines of Brassica napus were resynthesized via ovule culture through 24 interspecific crosses between four Brassica oleracea and three Brassica campestris accessions. The degree of success in the interspecific crosses was significantly influenced by maternal genotypes. The interspecific hybrid production rate (HPR) varied with combinations from 0 to 76.9%, with a mean HPR of 24.7% for the crosses with B. campestris as the female parent and 6.9% for the crosses with B. oleracea as female parent. Twenty‐four crosses between seven natural and six resynthesized B. napus gave, on average, 10.3 seeds per pod, and ranged from 1.2 to 22.0 seeds per pod, depending on genotypes of both parents. Resynthesized lines of B. napus showed high erucic acid content and variable content of linolenic acid, ranging from 3.4% to 9.9%. The fatty acid composition in hybrid seeds between natural and resynthesized B. napus was dominated by the embryo genotypes; an additive mode was shown for erucic acid and positive over‐dominance for linolenic acid content.     

Disease response of resynthesized Brassica napus L. lines carrying different combinations of resistance to Plasmodiophora brassicae Wor.

Resistance responses of resynthesized Brassica napus lines to infection with Plasmodiophora brassicae were investigated. Lines that were derived from interspecific crosses between clubroot-resistant B. rapa and resistant B. oleracea exhibited very broad and effective resistance in both greenhouse and field tests. When clubroot resistance was introduced into resynthesized lines from the B. oleracea parent only, the plants were mainly susceptible. Interspecific hybrids from the most resistant parental genotypes, i.e. B. campestris ECD-04 and the B. oleracea cultivars ECD-15 or ‘Bohmerwaldkohf’, were used to initiate a B. napus resistance-breeding programme. These artificial rapeseed lines were resistant to isolates that were virulent on all B. napus differential lines and/or parental lines. Preliminary segregation analysis suggests that their resistance is due to at least two dominant and unlinked genes. In some cases progenies from selfed resynthesized plants exhibited resistance reactions that differed from those of the parental hybrid plant; this may have been the result of cytological instability.     

Intergeneric hybrid between Brassica napus and Diplotaxis harra through ovary culture and the cytogenetic analysis of their progenies

Brassica napus (2n = 38) and Diplotaxis harra (2n = 26) were used to investigate gene transfer from D. harra to B. napus. Intergeneric F₁ hybrids (dihaploid 2n = 32 chromosomes) were obtained through ovary culture. The chromosome associations in the first meiotic division was (0–2)_III + (2–10)_II + (12–28)_I. Many seeds were harvested in the F₁ hybrid after backcrossing with B. napus, and from open pollination of the F₁ hybrid. Somatic chromosome numbers of BC₁ and hybrid plants varied from 2n = 26 to 52. In the first meiotic division, high frequencies of bivalent association and relatively low pollen fertility were observed. BC₂ plants generated from the BC₁ plants with 2n = 38 chromosomes, 69.6% showed 2n = 38 chromosomes. Many aneuploids with addition and deletion of chromosomes were also obtained. A bridge plant between B. napus and D. harra with 2n = 32 chromosomes should be valuable material for the breeding of brassica crops.     

Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences

Efficiencies of SCAR, CAPS and PCR-RF-SSCP marker production were investigated using two combinations of breeding lines in Brassica oleracea. Published EST sequences of B. oleracea, Brassica rapa, Brassica napus, and Arabidopsis thaliana and newly determined nucleotide sequences of anther cDNA clones from B. oleracea were used for designing primer pairs to amplify genes. The percentage of primer pairs yielding DNA amplification of a single gene was higher in primer pairs of B. oleracea (91%) than those of B. rapa (56%) and A. thaliana (17%). Single DNA fragments amplified by 9% of the primer pairs showed polymorphism as SCAR markers between a broccoli line and a Chinese kale line by agarose-gel electrophoresis. CAPS analysis showed different band patterns in 32% of the same-sized DNA fragments, and PCR-RF-SSCP analysis revealed DNA polymorphism in 52% of those showing no DNA polymorphism by CAPS. In total, 71% of the single DNA fragments were converted to DNA markers. The frequency of DNA polymorphism between parental lines of a cabbage F₁ hybrid was lower, 5% by SCAR and 12% by CAPS. However PCR-RF-SSCP analysis revealed DNA polymorphism in 21% of the DNA fragments showing no polymorphism by CAPS. These results suggest that PCR-RF-SSCP analysis enables highly efficient DNA marker production for mapping of genes in Brassica using progeny, even progeny of closely related parents. Analysis of selfed seeds of broccoli F₁ cultivars using PCR-RF-SSCP markers indicated that PCR-RF-SSCP analysis is also applicable to seed purity tests.     

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.     

Interspecific cross of Brassica oleracea var. alboglabra and B. napus : effects of growth condition and silique age on the efficiency of hybrid production, and inheritance of erucic acid in the self-pollinated backcross generation

Interspecific hybrids were produced from reciprocal crosses between Brassica napus (2n = 38, AACC) and B. oleracea var. alboglabra (2n = 18, CC) to introgress the zero-erucic acid alleles from B. napus into B. oleracea. The ovule culture embryo rescue technique was applied for production of F₁ plants. The effects of silique age, as measured by days after pollination (DAP), and growth condition (temperature) on the efficiency of this technique was investigated. The greatest numbers of hybrids per pollination were produced under 20°/15°C (day/night) at 16 DAP for B. oleracea (♀) × B. napus crosses, while under 15°/10°C at 14 DAP for B. napus (♀) × B. oleracea crosses. Application of the ovule culture technique also increased the efficiency of BC₁ (F₁ × B. oleracea) hybrid production by 10-fold over in vivo seed set. The segregation of erucic acid alleles in the self-pollinated backcross generation, i.e. in BC₁S₁ seeds, revealed that the gametes of the F₁ and BC₁ plants carrying a greater number of A-genome chromosomes were more viable. This resulted in a significantly greater number of intermediate and a smaller number of high-erucic acid BC₁S₁ seeds.