BackgroundAs seed oil content (OC) is a key measure of rapeseed quality, better understanding the genetic basis of OC would greatly facilitate the breeding of high-oil cultivars. Here, we investigated the components of genetic effects and genotype × environment interactions (GE) that govern OC using a full diallel set of nine parents, which represented a wide range of the Chinese rapeseed cultivars and pure lines with various OCs.ResultsOur results from an embryo-cytoplasm-maternal (GoCGm) model for diploid seeds showed that OC was primarily determined by genetic effects (VG) and GE (VGE), which together accounted for 86.19% of the phenotypic variance (VP). GE (VGE) alone accounted for 51.68% of the total genetic variance, indicating the importance of GE interaction for OC. Furthermore, maternal variance explained 75.03% of the total genetic variance, embryo and cytoplasmic effects accounted for 21.02% and 3.95%, respectively. We also found that the OC of F1 seeds was mainly determined by maternal effect and slightly affected by xenia. Thus, the OC of rapeseed was simultaneously affected by various genetic components, including maternal, embryo, cytoplasm, xenia and GE effects. In addition, general combining ability (GCA), specific combining ability (SCA), and maternal variance had significant influence on OC. The lines H2 and H1 were good general combiners, suggesting that they would be the best parental candidates for OC improvement. Crosses H3 × M2 and H1 × M3 exhibited significant SCA, suggesting their potentials in hybrid development.ConclusionsOur study thoroughly investigated and reliably quantified various genetic factors associated with OC of rapeseed by using a full diallel and backcross and reciprocal backcross. This findings lay a foundation for future genetic studies of OC and provide guidance for breeding of high-oil rapeseed cultivars.
A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus.
A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.
Self‐incompatibility (SI) is a genetic mechanism that rejects self‐pollen and thus prevents inbreeding in some hermaphroditic angiosperms. In the Brassicaceae, SI involves a pollen‐stigma recognition system controlled by a single locus known as the S locus, which consists of two highly polymorphic genes that encode S‐locus cysteine‐rich protein (SCR) and S‐receptor kinase (SRK). When self‐pollen lands on the stigma, the S‐haplotype‐specific interaction between SCR and SRK triggers SI. Here, we show that the GATA transcription factor BnA5.ZML1 suppresses SI responses in Brassica napus and is induced after compatible pollination. The loss‐of‐function mutant bna5.zml1 displays reduced self‐compatibility. In contrast, overexpression of BnA5.ZML1 in self‐incompatible stigmas leads to a partial breakdown of SI responses, suggesting that BnA5.ZML1 is a stigmatic compatibility factor. Furthermore, the expression levels of SRK and ARC1 are up‐regulated in bna5.zml1 mutants, and they are down‐regulated in BnA5.ZML1 overexpressing lines. SRK affects the cellular localization of BnA5.ZML1 through direct protein–protein interaction. Overall, our findings highlight the fundamental role of BnA5.ZML1 in SI responses in B. napus, establishing a direct interaction between BnA5.ZML1 and SRK in this process.
Summary Doubled haploid technology is widely used to accelerate plant breeding, but its use in the important oilseed crop Brassica napus L. is limited because B. napus haploids could only be obtained through in vitro anther or microspore cultures. Recently, maize (Zea mays) lines containing mutations in Domain of unknown function 679 membrane protein (DMP) were used as haploid inducer lines. This new haploid induction mechanism has been extended to several other plants, including the dicots Arabidopsis thaliana, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). Here, we knocked out four BnaDMP genes in the B. napus cultivar Westar using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 vector with an enhanced green fluorescent protein expression cassette. Plants with DMP mutations in B. napus in the T0, T1, and T2 generations exhibited a haploid induction rate up to 2.53%. These results suggest that targeting BnaDMP could be useful for haploid induction in B. napus.
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