Chromosome instabilities in resynthesized Brassica napus revealed by FISH.

Brassica napus is an allopolyploid plant, derived from spontaneous hybridization between Brassica rapa and Brassica oleracea. Intensive breeding has led to a significant reduction in genetic and phenotypic diversity within this species. Newly resynthesized hybrids from progenitor species may restore some diversity in B. napus, but they often are chromosomally and phenotypically unstable. Using fluorescence in situ hybridization, we tested chromosome constitutions in a range of new allopolyploids resynthesized from various parental species. A majority of these allopolyploids were euploid, with the expected chromosome numbers and constitutions, but deviations were also identified. We detected a low level of intergenomic rearrangements in analyzed hybrids and a high level of changes in rDNA loci. Our study revealed a significant effect of maternal cross combination on loss of 35S rDNA loci, especially when B. rapa was the maternal parent. The studied lines were characterized by diversified of pollen viability. In the analyzed hybrids, the erucic acid level in the seed oil ranged from 0 to 43.4% and total glucosinolate content in seeds ranged from 24.3 to 119.2 µmol g-1. Our study shows that cytogenetic analysis of B. napus resynthesized hybrids would be useful in breeding for the selection of lines with important agricultural characters and genetically stable stock seed production.

Estimation of seed yield in oilseed rape to identify the potential of semi-resynthesized parents for the development of new hybrid cultivars.

Resynthesized (RS) Brassica napus can be used to increase the genetic diversity of this important crop plant and to develop the heterotic gene pool required for successful hybrid breeding programmes. The level of heterosis in F1 hybrids depends on the individual performance of the parents and on the degree of genetic difference between them. However, RS forms obtained from crosses of B. rapa ssp. with B. oleracea ssp. possess many undesirable agronomic traits, such as low quality of seeds, low yield and seed oil content, high erucic acid level in the oil and high glucosinolate content in seed meal. Therefore, RS oilseed rape needs to be improved by crossing with natural double-low oilseed rape, leading to selected double-low quality semi-RS lines that can be used for breeding. In this study, we evaluated the seed yield potential of F1 hybrids derived from crosses between Ogura cytoplasmic male-sterility (CMS) lines and doubled haploid (DH) semi-RS lines with restorer gene in three locations in Poland. The genotype by environment interaction (GE interaction) and general combining ability (GCA) of the restorer and CMS line effects, as well as the effects of heterosis, were also assessed. The results of the study provide the first insights into the use of semi-RS lines as components for the development of new hybrid cultivars. Even the introduction of 50% of the RS oilseed rape genotype to natural restorer lines resulted in a marked heterosis effect, with seed yield ranging from 4.56% to 90.17% more than that of the better parent. The yield of the best hybrid amounted to 108.6% of the seed yield of the open-pollinated cultivar Monolit and 94.4% of that of the hybrid cultivar Arsenal. The best DH semi-RS line S1, which had a significantly positive GCA for seed yield, can be recommended as a possible parent for inclusion in breeding programmes aimed at developing new hybrid cultivars.

Genotype × environment interaction on tocochromanol and plastochromanol-8 content in seeds of doubled haploids obtained from F1 hybrid black × yellow seeds of winter oilseed rape (Brassica napus L.).

BACKGROUND: The aim of the study was to determine the effect of genotype × environment interaction on the levels of α-, ß-, γ- and δ-tocopherol (α-T, ß-T, γ-T and δ-T, respectively) and plastochromanol-8 (PC-8) in seeds of 17 doubled haploids (DHs) obtained from the F1 hybrid derived from crossing black (DH H2 -26) × yellow (DH Z-114) seeds of winter oilseed rape. RESULTS: The content of tocopherols in the tested DH lines ranged from 415.6 to 540.1 mg kg-1 seeds, while PC-8 content ranged from 56.3 to 89.0 mg kg-1 seeds. The α-T/γ-T ratio reached a level of 0.78-1.29. Studies have shown that heritability for α-T, ß-T, γ-T, total-T and PC-8 is mainly due to genotypic variation. For the δ-T homologue the level was dependent on environmental effect. CONCLUSION: The obtained DH lines population of oilseed rape is characterized by high heritability coefficients for α-T, ß-T, γ-T, total-T and PC-8 levels, which indicates a greater influence of genotype than the environment on the content of these compounds. Among all studied doubled haploids, seven DHs were selected which were characterized by stable contents of α-T, ß-T, γ-T, δ-T and total-T with the simultaneous stable content of PC-8. © 2017 Society of Chemical Industry.

Development of new restorer lines for CMS ogura system with the use of resynthesized oilseed rape (Brassica napus L.).

Resynthesized (RS) oilseed rape (Brassica napus L.) is potentially of great interest for hybrid breeding. However, a major problem with the direct use of RS B. napus is the quality of seed oil (high level of erucic acid) and seed meal (high glucosinolate content), which does not comply with double-low quality oilseed rape. Thus, additional developments are needed before RS B. napus can be introduced into breeding practice. In this study, RS oilseed rape was obtained through crosses between B. rapa ssp. chinensis var. chinensis and B. oleracea ssp. acephala var. sabellica. RS plant was then crossed with double-low (00) winter oilseed rape lines containing the Rfo gene for Ogura cytoplasmic male sterility (CMS ogu) system. Populations of doubled haploids (DH) were developed from these F1 hybrids using the microspore in vitro culture method. The seeds of semi-RS DH lines were analyzed for erucic acid and glucosinolate content. Among the populations of semi-RS DHs four 00-quality lines with the Rfo gene were selected. Using 344 AFLP markers to estimate genetic relatedness, we showed that the RS lines and semi-RS lines formed clusters that were clearly distinct from 96 winter oilseed rape parental lines of F1 hybrids.

Study of variation of tocochromanol and phytosterol contents in black and yellow seeds of Brassica napus L. doubled haploid populations.

BACKGROUND: In the study, an analysis of tocopherols, plastochomanol-8 and phytosterols was conducted using DH lines obtained from F1 hybrids of reciprocal crosses between yellow- and black-seeded lines. METHODS: The biological material for the study consisted of two DH populations of winter oilseed rape obtained from F1 hybrids of reciprocal crosses between two DH lines: yellow- and black-seeded. Seed color was determined using a ColorFlex spectrophotometer. Fat content was determined via pulsed NMR. The levels of tocopherols, and plastochromanol-8 are analyzed using HPLC. Phytosterol contents and composition were determined by the GC method. RESULTS: The fat content of the black-seeded parental line was 49% and this was higher than that of the yellow-seeded parental line (44%). The fat content of DH line populations ranged from 44 to 51%. Total tocopherol content ranged from 460 to 602 mg/kg and the α-T/γ-T ratio was from 0.66 to 1.09. In parental lines H2-26 and Z-114 the total tocopherol content was 534 and 525 mg/kg, but the α-T/γ-T ratios were 0.81 and 1.21, respectively. The yellow-seeded parental line (Z-114) was characterized by a higher PC-8 content (81 mg/kg) than the H2-26 black-seeded parental line (58 mg/kg). The largest part of the total phytosterol content in seeds of both populations was ß-sitosterol from 976 to 2148 mg/kg, followed by campasterol, from 636 to 1364 mg/kg, and brassicasterol from 375 to 678 mg/kg. The total tocopherol content ranged from 462 to 595 mg/kg (population HxZ) and from 460 to 602 mg/kg (population ZxH). Significantly positive correlations were observed between the seed color with α-T (r = 0.38, p < 0.01), γ-T (r = -0,34, p < 0.01) and PC-8 content (r = 0.29, p < 0.01). Correlations between the seed color with total tocopherol and total phytosterol content were not noted. CONCLUSIONS: Considering the range of genetic variation among doubled haploids of two populations, selected DH lines may be good parents for further breeding programs focused on increasing the amount and improving the quality of oilseed rapeseed oil. However, further studies will also be made to determine the influence of the environment on bioactive compounds in rapeseed oil. Cross direction of parental DH lines: yellow- and black-seeded has some influence, albeit not statistically significant, on the diversity of doubled haploid in their populations with regard to average fat, tocochromanol and phytosterol content.

Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress.

In this report we characterized the Arabidopsis ABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thaliana ABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.

Application of in vitro pollination of opened ovaries to obtain Brassica oleracea L. × B. rapa L. hybrids.

This study presents the results of experiments concerning: (1) interspecific hybridization of Brassica oleracea × Brassica rapa via application of in vitro placental pollination and (2) embryological analysis of the process of resynthesis of Brassica napus. In order to overcome certain stigma/style barriers, B. rapa pollen was placed in vitro on an opened B. oleracea ovary (with style removed). Pollinated ovaries were cultured on Murashige and Skoog (MS) medium. After 24-d culture, the developing embryos were isolated from immature seeds and transferred onto MS medium supplemented with 0.47 µM kinetin, 0.49 µM 1-naphthaleneacetic acid, and 10% (v/v) coconut water. When the embryos had turned green, they were immediately placed onto MS medium with 100 µM kinetin. After development of the seedling, plantlets were transferred to soil. Chromosome doubling was achieved after another week. Cytometric analysis of nuclear DNA confirmed the hybrid nature of the plants. Resynthesis of B. napus can be performed through interspecific hybridization of B. oleracea × B. rapa followed by embryo rescue and genome doubling.

Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) by using Agrobacterium tumefaciens.

Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by using Agrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing the uidA gene encoding beta-glucuronidase (GUS) and the bar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T1 plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.

Multivariate approach to evaluating the fatty acid composition of seed oil in a doubled haploid population of winter oilseed rape (Brassica napus L.).

The fatty acid composition of oil of the zero erucic acid commercial Brassica napus L. is typical for this species. It is rich in oleic acid and contains moderate levels of linoleic and linolenic acid. For human nutrition, it is advantageous primarily to obtain the highest possible content of oleic acid and to maintain the 2:1 ratio of linoleic to linolenic acid, while preserving the average total content of saturated acids. Uni- and multivariate analyses of variance were used for evaluation of doubled haploid lines of winter oilseed rape in respect of five fatty acids: palmitic (C16:0), stearic (C18:0) oleic (C18:1), linoleic (C18:2) and linolenic (C18:3). Some proposals of studying doubled haploid (DH) lines with the use of canonical transformation were also given. In MANOVA, the five original variables (individual fatty acids) were replaced by three 'new' variables (combinations of these acids) and used to evaluate DH lines with respect to the requirements concerning the nutritional role of fatty acids. The first variable was the total content of the saturated acids (C16:0 + C18:0), the second (unchanging) was the content of the monounsaturated acid C18:1, and the third was the difference between polyunsaturated acids, i.e. between linoleic acid, and the doubled content of linolenic acid (C18:2 - 2 x C18:3).