One of the goals in oilseed rape breeding is the identification of genotypes with low linolenic acid content in seed oil. Here, we present new genetic markers for mutant alleles of a chemically induced low linolenic rapeseed line DH219/05. Genomic clones comprising fatty acids desaturase 3 (FAD3) genes from the mutant and wild-type rapeseed lines were sequenced. Two statistically important single nucleotide polymorphisms were detected: (1) a C to T substitution in the third position of the sixth codon of the seventh exon in the BnaA.FAD3 gene and (2) a G to A transition in the 5¢ splice donor site of the sixth intron in the BnaC.FAD3 gene. Allele-specific SNP markers were designed involving detection of the wild-type and mutant alleles by SNaPshot analysis and locus-specific PCR primers. Strong negative correlation between the presence of mutant alleles in the A and C genomes and linolenic acid content was revealed by analysis of variance. Sequence analysis of transcript variants confirmed predictions on possible negative effects of mutations on FAD3 gene expression.
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.
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.
In this report we characterized the ArabidopsisABI1 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. thalianaABI1. 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0334-x) contains supplementary material, which is available to authorized users.
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 F 1 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 F 1 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.
Haploids and doubled haploids (DHs) have been produced in Brassica ssp. using anther or isolated microspore cultures. Since 1982, when Lichter developed a method of isolated microspores culture, this technique has been constantly improved and modified. Haploids and DH of Brassica napus have been extensively used in genetic studies, such as gene mapping, location of quantitative trait locus, marker / trait association studies, and genomics. Furthermore, the oilseed rape haploid induction technique can nowadays be efficiently combined with several other plant biotechnological techniques, enabling several novel breeding achievements, such as mutation breeding, the breeding of hybrid varieties, genetic transformation, and resynthesis of B. napus. The development and improvement of in vitro androgenesis of oilseed rape allowed DH populations to be obtained on a large scale. Today DH lines are used in the genetic analysis of quantitative traits and to analyze the impact of the environment on the yield and quality of the seeds. Currently, DH technology has become more efficient and hence can be widely applied in winter oilseed rape breeding. Additionally, DH technology is used to shorten the process of breeding new varieties. An open pollinated B. napus cv. Monolit (Plant Breeding Strzelce Ltd., Co.) is the first Polish winter oilseed rape variety obtained using DH technology. The breeding cycle of this variety is about four years shorter compared to a classical breeding program. The second Polish cultivar obtained using DH technology, cv. Brendy (Plant Breeding Smolice Ltd., Co.) was developed in a similarly short period. In the breeding of new oilseed rape hybrid varieties, homozygous restorer lines are also exploited. Our basic research used oilseed rape DH technology with a focus on improving the qualitative traits of oilseed rape and is presented here, based on the major researches conducted worldwide as well as on the results obtained at the Plant Breeding and Acclimati
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