Genome-wide characterization of ubiquitin-conjugating enzyme gene family explores its genetic effects on the oil content and yield of Brassica napus.

Ubiquitin-conjugating enzyme (UBC) is a critical part of the ubiquitin-proteasome pathway and plays crucial roles in growth, development and abiotic stress response in plants. Although UBC genes have been detected in several plant species, characterization of this gene family at the whole-genome level has not been conducted in Brassica napus. In the present study, 200 putative BnUBCs were identified in B. napus, which were clustered into 18 subgroups based on phylogenetic analysis. BnUBCs within each subgroup showed relatively conserved gene architectures and motifs. Moreover, the gene expression patterns in various tissues as well as the identification of cis-acting regulatory elements in BnUBC promoters suggested further investigation of their potential functions in plant growth and development. Furthermore, three BnUBCs were predicted as candidate genes for regulating agronomic traits related to oil content and yield through association mapping. In conclusion, this study provided a wealth of information on the UBC family in B. napus and revealed their effects on oil content and yield, which will aid future functional research and genetic breeding of B. napus.

A global survey of the transcriptome of allopolyploid Brassica napus based on single-molecule long-read isoform sequencing and Illumina-based RNA sequencing data.

Brassica napus is a recent allopolyploid derived from the hybridization of Brassica rapa (Ar Ar ) and Brassica oleracea (Co Co ). Because of the high sequence similarity between the An and Cn subgenomes, it is difficult to provide an accurate landscape of the whole transcriptome of B. napus. To overcome this problem, we applied a single-molecule long-read isoform sequencing (Iso-Seq) technique that can produce long reads to explore the complex transcriptome of B. napus at the isoform level. From the Iso-Seq data, we obtained 147 698 non-redundant isoforms, capturing 37 403 annotated genes. A total of 18.1% (14 934/82 367) of the multi-exonic genes showed alternative splicing (AS). In addition, we identified 549 long non-coding RNAs, the majority of which displayed tissue-specific expression profiles, and detected 7742 annotated genes that possessed isoforms containing alternative polyadenylation sites. Moreover, 31 591 AS events located in open reading frames (ORFs) lead to potential protein isoforms by in-frame or frameshift changes in the ORF. Illumina RNA sequencing of five tissues that were pooled for Iso-Seq was also performed and showed that 69% of the AS events were tissue-specific. Our data provide abundant transcriptome resources for a transcript isoform catalog of B. napus, which will facilitate genome reannotation, strengthen our understanding of the B. napus transcriptome and be applied for further functional genomic research.

A recessive high-density pod mutant resource of Brassica napus.

In Brassica napus, pod number and pod density are critical factors to determine seed yield. Although the pod density is an essential yield trait, the regulation of yield formation in oil crops, as well as the genetic and molecular mechanisms, are poorly understood. In this study, we characterized a rapeseed high-density pod mutant (dpt247) from composite hybridization. To shed some light on the nature of this mutation, it was investigated morphologically, anatomically, physiologically, genetically and transcriptomically. The mutant plant showed noticeable phenotypic differences in comparison with the control plant, including reduced plant height and primary branch length, decreased number of primary branches, significantly increased number of pod on the main inflorescence, and more compact pod distribution. Besides, the mutant had higher levels of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in the shoot apical meristem (SAM). The dense pod trait was controlled by two major recessive genes identified in the segregating genetic populations of GRE501 and dpt247. RNA sequencing indicated genes participated in auxin, cytokinin and WUS/CLV signalling pathway in dpt247 were more active in the mutant. These results provide important information for understanding the regulation of yield formation and high yield breeding in rapeseed.

Syntenic quantitative trait loci and genomic divergence for Sclerotinia resistance and flowering time in Brassica napus.

Oilseed rape (Brassica napus) is an allotetraploid with two subgenomes descended from a common ancestor. Accordingly, its genome contains syntenic regions with many duplicate genes, some of which may have retained their original functions, whereas others may have diverged. Here, we mapped quantitative trait loci (QTL) for stem rot resistance (SRR), a disease caused by the fungus Sclerotinia sclerotiorum, and flowering time (FT) in a recombinant inbred line population. The population was genotyped using B. napus 60K single nucleotide polymorphism arrays and phenotyped in six (FT) and nine (SSR) experimental conditions or environments. In total, we detected 30 SRR QTL and 22 FT QTL and show that some of the major QTL associated with these two traits were co-localized, suggesting a genetic linkage between them. Two SRR QTL on chromosome A2 and two on chromosome C2 were shown to be syntenic, suggesting the functional conservation of these regions. We used the syntenic properties of the genomic regions to exclude genes for selection candidates responsible for QTL-associated traits. For example, 152 of the 185 genes could be excluded from a syntenic A2-C2 region. These findings will help to elucidate polyploid genomics in future studies, in addition to providing useful information for B. napus breeding programs.

Gene Expression Changes During the Allo-/Deallopolyploidization Process of Brassica napus.

Gene expression changes due to allopolyploidization have been extensively studied in plants over the past few decades. Nearly all these studies focused on comparing the changes before and after genome merger. In this study, we used the uniquely restituted Brassica rapa (RBR, AeAe, 2n = 20) obtained from Brassica napus (AnAnCnCn, 2n = 38) to analyze the gene expression changes and its potential mechanism during the process of allo-/deallopolyploidization. RNA-seq-based transcriptome profiling identified a large number of differentially expressed genes (DEGs) between RBR and natural B. rapa (ArAr), suggesting potential effects of allopolyploidization/domestication of AA component of B. napus at the tetrapolyploid level. Meanwhile, it was revealed that up to 20% of gene expressions were immediately altered when compared with those in the An-subgenome. Interestingly, one fifth of these changes are in fact indicative of the recovery of antecedent gene expression alternations occurring since the origin of B. napus and showed association with homoeologous expression bias between An and Cn subgenomes. Enrichment of distinct gene ontology (GO) categories of the above sets of genes further indicated potential functional cooperation of the An and Cn subgenome of B. napus. Whole genome methylation analysis revealed a small number of DEGs were identified in the differentially methylated regions.

The high-quality genome of Brassica napus cultivar 'ZS11' reveals the introgression history in semi-winter morphotype.

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi-winter oilseed rape cultivar 'ZS11' and its comprehensive genomic comparison with the genomes of the winter-type cultivar 'Darmor-bzh' as well as two progenitors. The integrated BAC-to-BAC and whole-genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high-quality genome assembly of B. napus 'ZS11'. Within a short evolutionary period (~6700 years ago), semi-winter-type 'ZS11' and the winter-type 'Darmor-bzh' maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to 'Darmor-bzh', both two subgenomes of 'ZS11' are closely related to its progenitors, and the 'ZS11' genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi-winter-type 'ZS11' underwent potential genomic introgressions with B. rapa (Ar ). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization-responsive flowering time in 'ZS11' was first experienced HE, and then underwent genomic introgression event with Ar , which potentially has led to genetic differences in controlling vernalization in the semi-winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi-winter oilseed rape in Asia.

Vacuolar Iron Transporter BnMEB2 Is Involved in Enhancing Iron Tolerance of Brassica napus.

Iron toxicity is a nutrient disorder that severely affects crop development and yield in some soil conditions. Vacuolar detoxification of metal stress is an important strategy for plants to survive and adapt to this adverse environment. Vacuolar iron transporter (VIT) members are involved in this process and play essential roles in iron storage and transport. In this study, we identified a rapeseed VIT gene BnMEB2 (BnaC07g30170D) homologs to Arabidopsis MEB2 (At5g24290). Transient expression analysis revealed that BnMEB2 was localized to the vacuolar membrane. Q-PCR detection showed a high expression of BnMEB2 in mature (60-day-old) leaves and could be obviously induced by exogenous iron stress in both roots and leaves. Over-expressed BnMEB2 in both Arabidopsis wild type and meb2 mutant seedlings resulted in greatly improved iron tolerability with no significant changes in the expression level of other VIT genes. The mutant meb2 grew slowly and its root hair elongation was inhibited under high iron concentration condition while BnMEB2 over-expressed transgenic plants of the mutant restored the phenotypes with apparently higher iron storage in roots and dramatically increased iron content in the whole plant. Taken together, these results suggested that BnMEB2 was a VIT gene in rapeseed which was necessary for safe storage and vacuole detoxification function of excess iron to enhance the tolerance of iron toxicity. This research sheds light on a potentially new strategy for attenuating hazardous metal stress from environment and improving iron biofortification in Brassicaceae crops.

Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome.

Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes.

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis.

BACKGROUND: Sesame, Sesamum indicum L., is considered the queen of oilseeds for its high oil content and quality, and is grown widely in tropical and subtropical areas as an important source of oil and protein. However, the molecular biology of sesame is largely unexplored. RESULTS: Here, we report a high-quality genome sequence of sesame assembled de novo with a contig N50 of 52.2 kb and a scaffold N50 of 2.1 Mb, containing an estimated 27,148 genes. The results reveal novel, independent whole genome duplication and the absence of the Toll/interleukin-1 receptor domain in resistance genes. Candidate genes and oil biosynthetic pathways contributing to high oil content were discovered by comparative genomic and transcriptomic analyses. These revealed the expansion of type 1 lipid transfer genes by tandem duplication, the contraction of lipid degradation genes, and the differential expression of essential genes in the triacylglycerol biosynthesis pathway, particularly in the early stage of seed development. Resequencing data in 29 sesame accessions from 12 countries suggested that the high genetic diversity of lipid-related genes might be associated with the wide variation in oil content. Additionally, the results shed light on the pivotal stage of seed development, oil accumulation and potential key genes for sesamin production, an important pharmacological constituent of sesame. CONCLUSIONS: As an important species from the order Lamiales and a high oil crop, the sesame genome will facilitate future research on the evolution of eudicots, as well as the study of lipid biosynthesis and potential genetic improvement of sesame.

The genome of the mesopolyploid crop species Brassica rapa.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

Multiple source genes of HAmo SINE actively expanded and ongoing retroposition in cyprinid genomes relying on its partner LINE.

BACKGROUND: We recently characterized HAmo SINE and its partner LINE in silver carp and bighead carp based on hybridization capture of repetitive elements from digested genomic DNA in solution using a bead-probe 1. To reveal the distribution and evolutionary history of SINEs and LINEs in cyprinid genomes, we performed a multi-species search for HAmo SINE and its partner LINE using the bead-probe capture and internal-primer-SINE polymerase chain reaction (PCR) techniques. RESULTS: Sixty-seven full-size and 125 internal-SINE sequences (as well as 34 full-size and 9 internal sequences previously reported in bighead carp and silver carp) from 17 species of the family Cyprinidae were aligned as well as 14 new isolated HAmoL2 sequences. Four subfamilies (type I, II, III and IV), which were divided based on diagnostic nucleotides in the tRNA-unrelated region, expanded preferentially within a certain lineage or within the whole family of Cyprinidae as multiple active source genes. The copy numbers of HAmo SINEs were estimated to vary from 104 to 106 in cyprinid genomes by quantitative RT-PCR. Over one hundred type IV members were identified and characterized in the primitive cyprinid Danio rerio genome but only tens of sequences were found to be similar with type I, II and III since the type IV was the oldest subfamily and its members dispersed in almost all investigated cyprinid fishes. For determining the taxonomic distribution of HAmo SINE, inter-primer SINE PCR was conducted in other non-cyprinid fishes, the results shows that HAmo SINE- related sequences may disperse in other families of order Cypriniforms but absent in other orders of bony fishes: Siluriformes, Polypteriformes, Lepidosteiformes, Acipenseriformes and Osteoglossiforms. CONCLUSIONS: Depending on HAmo LINE2, multiple source genes (subfamilies) of HAmo SINE actively expanded and underwent retroposition in a certain lineage or within the whole family of Cyprinidae. From this perspective, HAmo SINE should provide useful phylogenetic makers for future analyses of the evolutionary relationships among species in the family Cyprinidae.

Sox genes evolution in closely related young tetraploid cyprinid fishes and their diploid relative.

Previous study and analysis of cytochrome b suggested that polyploidization event in the genus Tor occurred about 10 Mya ago. In order to understand evolutionary fates of Sox gene in the early stage of genome duplication at the nucleotide level, PCR surveys for Sox genes in three closely related cyprinid fishes T. douronensis (2n=100), T. qiaojiensis (2n=?), T. sinensis (2n=100) and their relative T. brevifilis (2n=50) were performed. Totally, 52 distinct Sox genes were obtained in these four species, representing SoxB, SoxC, and SoxE group. As expected, isoforms of some Sox genes correspond with the ploidy of species, such as two copies of Sox9a exist in tetraploid species. Analysis indicated that duplicated Sox gene pairs caused by polyploidization evolved independently of each other within polyploid species. Results of substitution rate showed nearly equal rate of nonsynonymous substitution of duplicated Sox orthologs among different polyploid species and their diploid relative orthologs, suggesting at the early stage of genome duplicated Sox orthologs are under similar selective constraints in different polyploidy species and their diploid relative at the amino acid level. All PCR fragments of Sox genes obtained in this study are not accompanied by obvious increase in mutations and pseudogene formation which means that they are under strong purifying selection, suggesting that they are functional at the DNA level. Genealogical analysis revealed that T. qiaojiensis was tetraploid, and T. douronensis, T. qiaojiensis as well as T. sinensis had an allotetraploid ancestor.