A new chromosome-scale genome of wild Brassica oleracea provides insights into the domestication of Brassica crops.

The cultivated diploid Brassica oleracea is an important vegetable crop, but the genetic basis of its domestication remains largely unclear in the absence of high-quality reference genomes of wild B. oleracea. Here, we report the first chromosome-level assembly of the wild Brassica oleracea L. W03 genome (total genome size, 630.7 Mb; scaffold N50, 64.6 Mb). Using the newly assembled W03 genome, we constructed a gene-based B. oleracea pangenome and identified 29 744 core genes, 23 306 dispensable genes, and 1896 private genes. We re-sequenced 53 accessions, representing six potential wild B. oleracea progenitor species. The results of the population genomic analysis showed that the wild B. oleracea populations had the highest level of diversity and represents the most closely related population to modern-day horticultural B. oleracea. In addition, the WUSCHEL gene was found to play a decisive role in domestication and to be involved in cauliflower and broccoli curd formation. We also illustrate the loss of disease-resistance genes during selection for domestication. Our results provide new insights into the domestication of B. oleracea and will facilitate the future genetic improvement of Brassica crops.

Genome-Wide Identification and Functional Characterization of Stress Related Glyoxalase Genes in Brassica napus L.

Rapeseed (Brassica napus L.) is not only one of the most important oil crops in the world, but it is also an important vegetable crop with a high value nutrients and metabolites. However, rapeseed is often severely damaged by adverse stresses, such as low temperature, pathogen infection and so on. Glyoxalase I (GLYI) and glyoxalase II (GLYII) are two enzymes responsible for the detoxification of a cytotoxic metabolite methylglyoxal (MG) into the nontoxic S-D-lactoylglutathione, which plays crucial roles in stress tolerance in plants. Considering the important roles of glyoxalases, the GLY gene families have been analyzed in higher plans, such as rice, soybean and Chinese cabbage; however, little is known about the presence, distribution, localizations and expression of glyoxalase genes in rapeseed, a young allotetraploid. In this study, a total of 35 BnaGLYI and 30 BnaGLYII genes were identified in the B. napus genome and were clustered into six and eight subfamilies, respectively. The classification, chromosomal distribution, gene structure and conserved motif were identified or predicted. BnaGLYI and BnaGLYII proteins were mainly localized in chloroplast and cytoplasm. By using publicly available RNA-seq data and a quantitative real-time PCR analysis (qRT-PCR), the expression profiling of these genes of different tissues was demonstrated in different developmental stages as well as under stresses. The results indicated that their expression profiles varied among different tissues. Some members are highly expressed in specific tissues, BnaGLYI11 and BnaGLYI27 expressed in flowers and germinating seed. At the same time, the two genes were significantly up-regulated under heat, cold and freezing stresses. Notably, a number of BnaGLY genes showed responses to Plasmodiophora brassicae infection. Overexpression of BnGLYI11 gene in Arabidopsis thaliana seedlings confirmed that this gene conferred freezing tolerance. This study provides insight of the BnaGLYI and BnaGLYII gene families in allotetraploid B. napus and their roles in stress resistance, and important information and gene resources for developing stress resistant vegetable and rapeseed oil.

Highly efficient preparation of active S-phenyl-L-cysteine with tryptophan synthase using a chemoenzymatic method.

BACKGROUND: S-Phenyl-L-cysteine is regarded as having potential applicability as an antiretroviral/protease inhibitor for human immunodeficiency virus (HIV). In the present study, optically active S-phenyl-L-cysteine was prepared in a highly efficient manner from inexpensive bromobenzene using tryptophan synthase through a chemoenzymatic method. RESULTS: The chemoenzymatic method used a four-step reaction sequence. The process started with the reaction of magnesium and bromobenzene, followed by a Grignard reaction, and then hydrolysis and enzymatic synthesis using tryptophan synthase. Through this approach, S-phenyl-L-cysteine was chemoenzymatically synthesized using tryptophan synthase from thiophenol and L-serine as the starting material. CONCLUSIONS: High-purity, optically active S-phenyl-L-cysteine was efficiently and inexpensively obtained in a total yield of 81.3% (> 99.9% purity).

High-throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus, Brassica rapa and Brassica oleracea.

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole-genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C-genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.

Protective effect of Rabdosia amethystoides (Benth) Hara extract on acute liver injury induced by Concanavalin A in mice through inhibition of TLR4-NF-κB signaling pathway.

Extract of Rabdosia amethystoides (Benth) Hara (ERA), a traditional Chinese medicine has antibacterial, antiviral, anti-tumor, anti-hepatitis and anti-inflammatory properties. However, the hepatoprotective effects and molecular mechanisms of ERA on acute liver injury have not been fully elucidated. This study aims to investigate the anti-inflammatory effect and liver protection of ERA against the acute liver injury induced by Concanavalin A (Con A) and its underlying molecular mechanisms in mice. Mice received ERA (50, 100, 150 mg/kg body weight) by gavage before Con A intravenous administration. We found that ERA pretreatment was able to significantly reduce the elevated serum alanine and aspartate aminotransferase levels and liver necrosis in Con A-induced hepatitis. In addition, ERA treatment significantly decreased the myeloperoxidase, malondialdehyde levels and augmented superoxide dismutase level in the liver tissue, and also suppressed the secretion of proinflammatory cytokines in the serum, compared with Con A group by enzyme linked immunosorbent assay. Furthermore, we observed that ERA pretreatment can significantly decrease the expression level of Toll-like receptor (TLR) 4 mRNA or protein in liver tissues. Further results showed that ERA pretreatment was capable of attenuating the activation of the NF-κB pathway by inhibiting IκBα kinase and p65 phosphorylation in Con A-induced liver injury. Our results demonstrate that ERA pretreatment has hepatoprotective property against Con A-induced liver injury through inhibition of inflammatory mediators in mice. The beneficial effect of ERA may be mediated by the downregulation of TLR4 expression and the inhibition of NF-κB activation.

Characterization of FAE1 in the zero erucic acid germplasm of Brassica rapa L.

The modification of erucic acid content in seeds is one of the major goals for quality breeding in oil-yielding Brassica species. However, few low erucic acid (LEA) resources are available, and novel LEA genetic resources are being sought. Fatty acid elongase 1 (FAE1) is the key gene that controls erucic acid synthesis. However, the mechanism for erucic acid synthesis in B. rapa lacks systematic study. Here, we isolated zero erucic acid lines from 1981 Chinese landraces of B. rapa and found that the formation of LEA is not attributable to variations in FAE1 coding sequences, as reported for B. napus, but may be attributable to the decrease in FAE1 expression. Moreover, the FAE1 promoter sequences of LEA and high erucic acid materials shared 95% similarity. Twenty-eight bases deletions (containing a 24-base AT-rich region) were identified approximately 1300 bp upstream from the FAE1 start codon in the LEA accessions. The genotype with the deletions co-segregated with the LEA trait in the segregating population. This study isolated an LEA B. rapa resource that can be exploited in Brassica cultivation. The promoter variations might modify the expression level of FAE1, and the results shed light on novel regulation mechanisms for erucic acid synthesis.

Perfluorononanoic acid disturbed the metabolism of lipid in the liver of streptozotocin-induced diabetic rats.

Most studies on the liver toxicity of perfluorinated compounds (PFCs) are focused on healthy individuals, whereas the effects of PFCs on individuals with diabetes mellitus have not been fully characterized. This study aimed to investigate the acute exposure of perfluorononanoic acid (PFNA) on the metabolism of lipid in the liver of streptozotocin-induced diabetic rats. Male diabetic rats were orally dosed by gavage for 7 days with 0, 0.2, 1 and 5 mg/kg/day PFNA. The contents of lipid, the activities of enzyme, the expressions of protein in the liver and the serum parameters were detected. The results indicate that dose-dependent accumulation of triglyceride and total cholesterol occurred in the livers of diabetic rats after PFNA treatment. PFNA increased the activities of lipid synthetase, fatty acid synthease, glucose-6-phosphate dehydrogenase and decreased the activity of lipolytic enzyme, hepatic lipase, in the liver of diabetic rats. The changes of the isocitrate dehydrogenase, malicenzyme and lipoprotein lipase were not obvious. The expressions of protein related to lipid homeostasis, liver X receptor α and apolipoprotein E, were decreased after PFNA administration. Exposure to PFNA also increased the activity of serum alanine aminotransferase in diabetic rats. In conclusion, this study discloses that exposure to PFNA impacts on enzymes and proteins related to liver lipid metabolism and lead to obvious accumulation of lipid in the liver of diabetic rats, which may be responsible for hepatotoxicity of this compound in individuals with diabetes mellitus.

Genome-wide investigation of genetic changes during modern breeding of Brassica napus.

KEY MESSAGE: Considerable genome variation had been incorporated within rapeseed breeding programs over past decades. In past decades, there have been substantial changes in phenotypic properties of rapeseed as a result of extensive breeding effort. Uncovering the underlying patterns of allelic variation in the context of genome organisation would provide knowledge to guide future genetic improvement. We assessed genome-wide genetic changes, including population structure, genetic relatedness, the extent of linkage disequilibrium, nucleotide diversity and genetic differentiation based on F ST outlier detection, for a panel of 472 Brassica napus inbred accessions using a 60 k Brassica Infinium® SNP array. We found genetic diversity varied in different sub-groups. Moreover, the genetic diversity increased from 1950 to 1980 and then remained at a similar level in China and Europe. We also found ~6-10 % genomic regions revealed high F ST values. Some QTLs previously associated with important agronomic traits overlapped with these regions. Overall, the B. napus C genome was found to have more high F ST signals than the A genome, and we concluded that the C genome may contribute more valuable alleles to generate elite traits. The results of this study indicate that considerable genome variation had been incorporated within rapeseed breeding programs over past decades. These results also contribute to understanding the impact of rapeseed improvement on available genome variation and the potential for dissecting complex agronomic traits.

Comparison of the heat stress induced variations in DNA methylation between heat-tolerant and heat-sensitive rapeseed seedlings.

DNA methylation is responsive to various biotic and abiotic stresses. Heat stress is a serious threat to crop growth and development worldwide. Heat stress results in an array of morphological, physiological and biochemical changes in plants. The relationship between DNA methylation and heat stress in crops is relatively unknown. We investigated the differences in methylation levels and changes in the cytosine methylation patterns in seedlings of two rapeseed genotypes (heat-sensitive and heat-tolerant) under heat stress. Our results revealed that the methylation levels were different between a heat-tolerant genotype and a heat-sensitive one under control conditions. Under heat treatment, methylation increased more in the heat-sensitive genotype than in the heat-tolerant genotype. More DNA demethylation events occurred in the heat-tolerant genotype, while more DNA methylation occurred in the heat-sensitive genotype. A large and diverse set of genes were affected by heat stress via cytosine methylation changes, suggesting that these genes likely play important roles in the response and adaption to heat stress in Brassica napus L. This study indicated that the changes in DNA methylation differed between heat-tolerant and heat-sensitive genotypes of B. napus in response to heat stress, which further illuminates the molecular mechanisms of the adaption to heat stress in B. napus.

Assessing and broadening genetic diversity of a rapeseed germplasm collection.

Assessing the level of genetic diversity within a germplasm collection contributes to evaluating the potential for its utilization as a gene pool to improve the performance of cultivars. In this study, 45 high-quality simple sequence repeat (SSR) markers were screened and used to estimate the genetic base of a world-wide collection of 248 rapeseed (Brassica napus) inbred lines. For the whole collection, the genetic diversity of A genome was higher than that of C genome. The genetic diversity of C genome for the semi-winter type was the lowest among the different germplasm types. Because B. oleracea is usually used to broaden the genetic diversity of C genome in rapeseed, we evaluated the potential of 25 wild B. oleracea lines. More allelic variations and a higher genetic diversity were observed in B. oleracea than in rapeseed. One B. oleracea line and one oilseed B. rapa line were used to generate a resynthesized Brassica napus line, which was then crossed with six semi-winter rapeseed cultivars to produce 7 F1 hybrids. Not only the allele introgression but also mutations were observed in the hybrids, resulting in significant improvement of the genetic base.