Intergeneric (intersubtribe) hybridization between Moricandia arvensis and Brassica A and B genome species by ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (C₃–C₄ intermediate species) and Brassica A and B genome species (B. campestris and B. nigra) were produced via ovary culture. When M. arvensis was used as a female parent, the hybrid embryo yield (0.25–0.45 embryo per pollination) was similar between two genomes, regardless of the male parent. The reciprocal hybrid using B. campestris as a female was also obtained, although yield of embryo was lower (0.02 embryo per pollination). On the other hand, no hybrids were obtained without the in vitro technique. As most hybrid embryos could not develop normal shoots, plants were regenerated by inducing shoots on the cultured hypocotyl. The hybrid nature of the regenerated plant was confirmed morphologically and cytogenetically. A certain amount of bivalents (2.52-2.71) in the hybrids indicated the existence of partial chromosome homology between two genera. The present results indicate that ovary culture is an effective technique for overcoming the crossing barrier between M. arvensis and Brassica cultivated species.     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Intergeneric hybridization between Hordeum and Hordelymus (Poaceae)

Intergeneric hybridization between the monotypic Eurasian genus Hordelymus and species of Hordeum was performed. Hybrids were obtained in nine combinations and the meiotic pairing was analyzed in four combinations (6 families). Hordelymus europaeus is probably an alloploid with two different genomes. The very low pairing in the hybrids with H. jubatum, H. brachyantherum , and H. pusillum indicates that these species do not have any genomes in common with Hordelymus. The hybrids with H. depressum have an intermediate pairing which suggests that there is a certain homoeology in one genome.     

Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) O.E. Schulz

The frequency of gene flow from Brassica napus L. (canola) to four wild relatives, Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L. and Erucastrum gallicum (Willd.) O.E. Schulz, was assessed in greenhouse and/or field experiments, and actual rates measured in commercial fields in Canada. Various marker systems were used to detect hybrid individuals: herbicide resistance traits (HR), green fluorescent protein marker (GFP), species-specific amplified fragment length polymorphisms (AFLPs) and ploidy level. Hybridization between B. rapa and B. napus occurred in two field experiments (frequency approximately 7%) and in wild populations in commercial fields (approximately 13.6%). The higher frequency in commercial fields was most likely due to greater distance between B. rapa plants. All F(1) hybrids were morphologically similar to B. rapa, had B. napus- and B. rapa-specific AFLP markers and were triploid (AAC, 2n=29 chromosomes). They had reduced pollen viability (about 55%) and segregated for both self-incompatible and self-compatible individuals (the latter being a B. napus trait). In contrast, gene flow between R. raphanistrum and B. napus was very rare. A single R. raphanistrum x B. napus F1 hybrid was detected in 32,821 seedlings from the HR B. napus field experiment. The hybrid was morphologically similar to R. raphanistrum except for the presence of valves, a B. napus trait, in the distorted seed pods. It had a genomic structure consistent with the fusion of an unreduced gamete of R. raphanistrum and a reduced gamete of B. napus (RrRrAC, 2n=37), both B. napus- and R. raphanistrum-specific AFLP markers, and had <1% pollen viability. No hybrids were detected in the greenhouse experiments (1,534 seedlings), the GFP field experiment (4,059 seedlings) or in commercial fields in Québec and Alberta (22,114 seedlings). No S. arvensis or E. gallicum x B. napus hybrids were detected (42,828 and 21,841 seedlings, respectively) from commercial fields in Saskatchewan. These findings suggest that the probability of gene flow from transgenic B. napus to R. raphanistrum, S. arvensis or E. gallicum is very low (<2-5 x 10(-5)). However, transgenes can disperse in the environment via wild B. rapa in eastern Canada and possibly via commercial B. rapa volunteers in western Canada.     

Genetic diversity of allozymes in turnip (Brassica rapa L. var. rapa) from the Nordic area

Genetic diversity and relationships based on isozymes were studied in 31 accessions of turnip (Brassica rapa L. var. rapa). The material included varieties, elite stocks, landraces and older turnip of slash-and-burn type from the Nordic area. A total of 9 isozyme loci and 26 alleles were studied. The isozyme systems were ACO, DIA, GPI, GOT, PGM, PGD and SKD. The level of heterozygosity was reduced in the landraces, but it was high for the variety group 'Ostersundom'. Turnip has a higher genetic variation than other crops within B. rapa and than in other species with the same breeding system. The genetic diversity showed that 18.7% of the genetic variation was within the accessions, and the total H tau value was 0.358. Gpi-I and Pgd-I showed the lowest variation compared with the other loci. The cluster analysis revealed five clusters, with one main cluster including 25 of the 31 accessions. The dendrogram indicated that the variety group 'Ostersundom' clustered together whereas the variety group 'Bortfelder' was associated with country of origin. The landraces were spread in different clusters. The 'slash-and-burn' type of turnip belonged to two groups.     

Progressive introgression between Brassica napus (oilseed rape) and B. rapa

We have earlier shown extensive introgression between oilseed rape (Brassica napus) and B. rapa in a weedy population using AFLP markers specific for the nuclear genomes. In order to describe the progress of this introgression, we examined 117 offspring from 12 maternal plants from the introgressed population with the same AFLP-markers; AFLP data were supported by chromosome counting. We also analysed the offspring with a species-specific chloroplast marker and finally evaluated the reproductive system in selected maternal plants. Our results indicated a high outcrossing rate of the introgressed maternal plants. It seemed that B. rapa most often functioned as the maternal plant in the introgression process and that the amount of oilseed rape DNA was highly diminished in the offspring compared to their introgressed maternal plants. However, our analysis of plants from the weedy population indicated that introgression can lead to both (1) exchange of chloroplast DNA between species producing B. rapa-like plants with B. napus chloroplasts and (2) incorporation of B. napus C-genome DNA into the B. rapa genome. Therefore, we question whether it can be regarded as containment to position transgenes in the chloroplast or in specific parts of the nuclear genome of B. napus.     

Integration of Brassica A genome genetic linkage map between Brassica napus and B. rapa.

An integrated linkage map between B. napus and B. rapa was constructed based on a total of 44 common markers comprising 41 SSR (33 BRMS, 6 Saskatoon, and 2 BBSRC) and 3 SNP/indel markers. Between 3 and 7 common markers were mapped onto each of the linkage groups A1 to A10. The position and order of most common markers revealed a high level of colinearity between species, although two small regions on A4, A5, and A10 revealed apparent local inversions between them. These results indicate that the A genome of Brassica has retained a high degree of colinearity between species, despite each species having evolved independently after the integration of the A and C genomes in the amphidiploid state. Our results provide a genetic integration of the Brassica A genome between B. napus and B. rapa. As the analysis employed sequence-based molecular markers, the information will accelerate the exploitation of the B. rapa genome sequence for the improvement of oilseed rape.     

Fitness of hybrids between rapeseed (Brassica napus) and wild Brassica rapa in natural habitats

Fitness of hybrids between genetically modified (GM) crops and wild relatives influences the likelihood of ecological harm. We measured fitness components in spontaneous (non-GM) rapeseed x Brassica rapa hybrids in natural populations. The F1 hybrids yielded 46.9% seed output of B. rapa, were 16.9% as effective as males on B. rapa and exhibited increased self-pollination. Assuming 100% GM rapeseed cultivation, we conservatively predict < 7000 second-generation transgenic hybrids annually in the United Kingdom (i.e. approximately 20% of F1 hybrids). Conversely, whilst reduced hybrid fitness improves feasibility of bio-containment, stage projection matrices suggests broad scope for some transgenes to offset this effect by enhancing fitness.     

Genetic analysis of novel intra-species unilateral incompatibility in Brassica rapa (syn. campestris) L.

Plants have evolved many systems to prevent inappropriate fertilization. Among them, incompatibility is a well-organized system in which pollen germination or pollen-tube growth is inhibited in pistils. Self-incompatibility (SI), rejecting self-pollen, promotes outbreeding in flowering plants. On the other hand, inter-species incompatibility, preventing gene flow among species to restrict outbreeding, usually occurs unilaterally, and is known as unilateral incompatibility (UI). In Brassicaceae, little is known about the molecular mechanism of UI, although S-locus genes involved in recognition of self-pollen have been characterized in the SI system. In the present study, we characterized novel UI observed between members of the same species, Brassica rapa; pollen of Turkish SI lines was specifically rejected by pistils of the Japanese commercial SI variety Osome. The incompatible phenotype of this intra-species UI closely resembled that of SI. Segregation analysis revealed that the pollen factor of this UI was not linked to the S-locus.The revised version was published online in December 2004 with corrections to figure 1.     

Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa

It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process. Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events, and ranged from 7.6×10⁵ to 13.8×10⁵ (counts per second) in contrast with the wild-type at 5.3×10⁵ cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic canola parents. However, fluorescence intensities of the hemizygous F₁ hybrid lines were lower than the respective original homozygous canola parents. Each F₁ hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used as a tool to monitor transgene flow from crop species to wild relatives. Received: 11 September 2000 / Accepted: 1 February 2001     

Intergeneric hybridization between Brassica napus and Sinapis pubescens,and the cytology and crossability of their progenies

The cytological possibility of gene transfer from Sinapis pubescens to Brassica napus was investigated. Intergeneric hybrids between Brassica napus (2n = 38) and Sinapis pubescens (2n = 18) were produced through ovary culture. The F₁ hybrids were dihaploid and the chromosome configurations were (0–1) _III + (2–11) _II + (5–24) _I . One F₂ plant with 38 chromosomes was obtained from open pollination of the F₁ hybrid. Thirty-one seeds were obtained from the backcross of the F₂ plant with B. napus. Five out of seven plants had 38 chromosomes, and the pollen stainability ranged from 0% to 81.4%. In the B₂ plants obtained from the backcross of B₁ plants with B. napus, 66.7% of the plants examined had 38 chromosomes. S. pubescens may become a gene source for the improvement of B. napus.     

Frequency-dependent fitness of hybrids between oilseed rape (Brassica napus) and weedy B. rapa (Brassicaceae)

Fitness of interspecific hybrids is sometimes high relative to their parents, despite the conventional belief that they are mostly unfit. F(1) hybrids between oilseed rape (Brassica napus) and weedy B. rapa can be significantly more fit than their weedy parents under some conditions; however, under other conditions they are less fit. To understand the reasons, we measured the seed production of B. napus, B. rapa, and different generations of hybrid plants at three different densities and in mixtures of different frequencies (including pure stands). Brassica napus, B. rapa, and backcross plants (F(1) ♀ × B. rapa) produced many more seeds per plant in pure plots than in mixtures and more seeds in plots when each was present at high frequency. The opposite was true for F(1) plants that produced many more seeds than B. rapa in mixtures, but fewer in pure stands. Both vegetative and reproductive interactions may be responsible for these effects. Our results show that the fitness of both parents and hybrids is strongly frequency-dependent and that the likelihood of introgression of genes between the species thus may depend on the numbers and densities of parents and their various hybrid offspring in the population.     

Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.     

Intergeneric hybridization and C-banding patterns inHordelymus (Triticeae,Poaceae)

Crosses ofHordelymus europaeus (2n = 4x = 28) with four genera in theTriticeae were attempted. Adult hybrids were obtained in combinations withHordeum bogdanii (2x),Hordeum depressum (4x), andSecale cereale (2x). The meiotic pairing was very low in the hybrids withH. bogdanii andSecale cereale (0.12 and 0.30 chiasmata/cell, respectively), whereas high pairing (9.90 chiasmata/cell) was found in hybrids withH. depressum due to autosyndetic pairing ofH. depressum chromosomes. The chromosome complement ofHordelymus europaeus comprised 16 metacentrics, 8 submetacentrics, and 4 SAT-chromosomes. The Giemsa C-banding patterns of the chromosomes were characterized by small to minute bands at no preferential positions. It is hypothesized thatHordelymus europaeus may genomically be closest related toTaeniatherum andPsathyrostachys spp.     

Intergeneric hybridization between Hordeum and Secale (Poaceae). I. Crosses and development of hybrids

A total of 241 intergeneric combinations between Hordeum and Secale were attempted. It was only possible to obtain hybrid offspring from combinations with Hordeum as the female parent. By the aid of embryo culture technique 81 intergeneric combinations were synthesized, 72 of them not reported earlier. Generally hybridization with the polyploid species of Hordeum was most successful. Morphologically the hybrids were intermediate to the parents in most characters, though an obvious effect of genome dosage could be observed. Only few deviations from the expected chromosome numbers of the hybrids were found. All hybrid plants showed complete male sterility.     

Genetic relatedness between streptomycin-producing and non-producing strains of Streptomyces griseus, studied by means of DNA-DNA hybridization.

DNA-DNA hybridization was studied in order to determine the genetic relatedness between a streptomycin-producing and a non-producing mutant of Streptomyces griseus. The latter strain of short vegetative life cycle had been developed from the streptomycin-producing strain by means of nitrogen mustard treatment. Since the two strains differ in several features, we were prompted to examine the sequence homology between their DNAs. Hybridization experiments carried out with the membrane filter method yielded 73.8% and 67.3% DNA homology values, respectively. The results indicate that there is a significant, but not very close, relatedness between the DNA sequences that corresponds to the similarities and differences observed between the phenotypes of the two strains. Further aspects of established homology are also discussed.