HO-CR and HOLL-CR: new forms of winter oilseed rape (Brassica napus L.) with altered fatty acid composition and resistance to selected pathotypes of Plasmodiophora brassicae (clubroot).

The priority in oilseed rape (Brassica napus L.) research and breeding programs worldwide is to combine different features to develop cultivars tailored to specific applications of this crop. In this study, forms with a modified fatty acid composition of seed oil were successfully combined with a source of resistance to Plasmodiophora brassicae Wor., a harmful protist-causing clubroot. Three HO-type recombinants in F6-F12 generations with oleic acid content of 80.2-82.1% and one HOLL-type F6 inbred mutant recombinant (HOmut × LLmut), with a high oleic acid content (80.9%) and reduced linolenic acid content (2.3%), were crossed with the cultivar Tosca, resistant to several pathotypes of P. brassicae. The work involved genotyping with the use of DNA markers specific for allelic variants of desaturase genes responsible for the synthesis of oleic and linolenic fatty acids, CAPS (FAD2 desaturase, C18:1), and SNaPshot (FAD3 desaturase, C18:3), respectively. Of 350 progenies in the F3 generation, 192 (55%) were selected for further studies. Among them, 80 HO (≥ 72%) lines were identified, 10 of which showed resistance to at least one up to four P. brassicae pathotypes. Thirty lines in the selected progeny contained high oleic acid and less than 5% linolenic acid; eight of them belonged to the HOLL type conferring resistance to at least one pathotype. Two HO lines and two HOLL lines were resistant to four pathotypes. The resulting HO-CR and HOLL-CR inbred lines with altered seed oil fatty acid composition and resistance to P. brassicae represent unique oilseed rape material with the desired combination of valuable traits.

QTL Genetic Mapping Study for Traits Affecting Meal Quality in Winter Oilseed Rape (Brassica Napus L.).

Rapeseed (Brassica napus L.) meal is an important source of protein, but the presence of anti-nutritional compounds, such as fibre and glucosinolates, still limits its use as a livestock feed. Understanding the genetic basis of seed fibre biosynthesis would help to manipulate its content in seeds of oilseed rape. Here, we applied high-resolution skim genotyping by sequencing (SkimGBS) and characterised 187,835 single-nucleotide polymorphism (SNP) markers across a mapping population subsequently used for a genetic mapping study (R/qtl). This approach allowed the identification of 11 stable QTL related to seed quality traits and led to the identification of potential functional genes underlying these traits. Among these, key genes with a known role in carbohydrate metabolic process, cell wall, lignin, and flavonoid biosynthesis, including cellulase GH5, TT10/LAC15, TT4, and SUC2, were found. This study furthers the understanding of the molecular mechanisms underlying seed fibre content and provides new markers for molecular breeding in B. napus.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene.

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low ("00") cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new "00" varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5'-C↓TAG-3' sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Marker assisted selection of new high oleic and low linolenic winter oilseed rape (Brassica napus L.) inbred lines revealing good agricultural value.

Development of oilseed rape (Brassica napus L.) breeding lines producing oil characterized by high oleic and low linolenic acid content is an important goal of rapeseed breeding programs worldwide. Such kind of oil is ideal for deep frying and can also be used as a raw material for biodiesel production. By performing chemical mutagenesis using ethyl methanesulfonate, we obtained mutant winter rapeseed breeding lines that can produce oil with a high content of oleic acid (C18:1, more than 75%) and a low content of linolenic acid (C18:3, less than 3%). However, the mutant lines revealed low agricultural value as they were characterized by low seed yield, low wintering, and high content of glucosinolates in seed meal. The aim of this work was to improve the mutant lines and develop high-oleic and low-linolenic recombinants exhibiting both good oil quality and high agronomic value. The plant materials used in this study included high-oleic and low-linolenic mutant breeding lines and high-yielding domestic canola-type breeding lines of good agricultural value with high oleic acid content and extremely low glucosinolates content. Field trials were conducted in four environments, in a randomized complete block design. Phenotyping was performed for wintering, yield of seed and oil, and seed quality traits. Genotype × environment interaction was investigated with respect to the content of C18:1 and C18:3 acids in seed oil. Genotyping was done for the selection of homozygous high oleic and low linolenic lines using allele-specific CAPS markers and SNaPshot assay, respectively. Finally, new high oleic and low linolenic winter rapeseed recombinant lines were obtained for use as a starting material for the development of new varieties that may be of high value on the oil crop market.

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.