Comparative Analysis between Homoeologous Genome Segments of Brassica napus and Its Progenitor Species Reveals Extensive Sequence-Level Divergence

Homoeologous regions of Brassica genomes were analyzed at the sequence level. These represent segments of the Brassica A genome as found in Brassica rapa and Brassica napus and the corresponding segments of the Brassica C genome as found in Brassica oleracea and B. napus. Analysis of synonymous base substitution rates within modeled genes revealed a relatively broad range of times (0.12 to 1.37 million years ago) since the divergence of orthologous genome segments as represented in B. napus and the diploid species. Similar, and consistent, ranges were also identified for single nucleotide polymorphism and insertion-deletion variation. Genes conserved across the Brassica genomes and the homoeologous segments of the genome of Arabidopsis thaliana showed almost perfect collinearity. Numerous examples of apparent transduplication of gene fragments, as previously reported in B. oleracea, were observed in B. rapa and B. napus, indicating that this phenomenon is widespread in Brassica species. In the majority of the regions studied, the C genome segments were expanded in size relative to their A genome counterparts. The considerable variation that we observed, even between the different versions of the same Brassica genome, for gene fragments and annotated putative genes suggest that the concept of the pan-genome might be particularly appropriate when considering Brassica genomes.     

Sequence‐level comparative analysis of the Brassica napus genome around two stearoyl‐ACP desaturase loci

We conducted a sequence‐level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl‐ACP desaturase‐encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (～56%) is lower than has been reported previously between A. thaliana and Brassica (～66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 ± 3.9% and 85.8 ± 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 ± 3.1% between the B. napus A genome and B. rapa, and 93.1 ± 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.     

Gene Silencing of BnTT10 Family Genes Causes Retarded Pigmentation and Lignin Reduction in the Seed Coat of Brassica napus

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.     

Promoter Variation and Transcript Divergence in Brassicaceae Lineages of FLOWERING LOCUS T

Brassica napus (AACC, 2n = 38), an oil crop of world-wide importance, originated from interspecific hybridization of B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18), and has six FLOWERING LOCUS T (FT) paralogues. Two located on the homeologous chromosomes A2 and C2 arose from a lineage distinct from four located on A7 and C6. A set of three conserved blocks A, B and C, which were found to be essential for FT activation by CONSTANS (CO) in Arabidopsis, was identified within the FT upstream region in B. napus and its progenitor diploids. However, on chromosome C2, insertion of a DNA transposable element (TE) and a retro-element in FT upstream blocks A and B contributed to significant structural divergence between the A and C genome orthologues. Phylogenetic analysis of upstream block A indicated the conserved evolutionary relationships of distinct FT genes within Brassicaceae. We conclude that the ancient At-α whole genome duplication contributed to distinct ancestral lineages for this key adaptive gene, which co-exist within the same genus. FT-A2 was found to be transcribed in all leaf samples from different developmental stages in both B. rapa and B. napus, whereas FT-C2 was not transcribed in either B. napus or B. oleracea. Silencing of FT-C2 appeared to result from TE insertion and consequent high levels of cytosine methylation in TE sequences within upstream block A. Interestingly, FT-A7/C6 paralogues were specifically silenced in winter type B. napus but abundantly expressed in spring type cultivars under vernalization-free conditions. Motif prediction indicated the presence of two CO protein binding sites within all Brassica block A and additional sites for FT activation in block C. We propose that the ancestral whole genome duplications have contributed to more complex mechanisms of floral regulation and niche adaptation in Brassica compared to Arabidopsis.     

The Tarenaya hassleriana Genome Provides Insight into Reproductive Trait and Genome Evolution of Crucifers

The Brassicaceae, including Arabidopsis thaliana and Brassica crops, is unmatched among plants in its wealth of genomic and functional molecular data and has long served as a model for understanding gene, genome, and trait evolution. However, genome information from a phylogenetic outgroup that is essential for inferring directionality of evolutionary change has been lacking. We therefore sequenced the genome of the spider flower (Tarenaya hassleriana) from the Brassicaceae sister family, the Cleomaceae. By comparative analysis of the two lineages, we show that genome evolution following ancient polyploidy and gene duplication events affect reproductively important traits. We found an ancient genome triplication in Tarenaya (Th-α) that is independent of the Brassicaceae-specific duplication (At-α) and nested Brassica (Br-α) triplication. To showcase the potential of sister lineage genome analysis, we investigated the state of floral developmental genes and show Brassica retains twice as many floral MADS (for MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUM RESPONSE FACTOR) genes as Tarenaya that likely contribute to morphological diversity in Brassica. We also performed synteny analysis of gene families that confer self-incompatibility in Brassicaceae and found that the critical SERINE RECEPTOR KINASE receptor gene is derived from a lineage-specific tandem duplication. The T. hassleriana genome will facilitate future research toward elucidating the evolutionary history of Brassicaceae genomes.     

A computational study of elongation factor G (EFG) duplicated genes: diverged nature underlying the innovation on the same structural template

Background

Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG''s evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function.

Methodology/Principal Findings

We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif “RGITI”; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV.

Conclusions/Significance

Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes.     

Expression profiles and genomic organisation of group A protein phosphatase 2C genes in Brassica oleracea

We investigated the expression profiles and genomic organisation of the ABA‐responsive genes encoding protein phosphatases 2C (PP2C, group A members) in Brassica oleracea to better understand their functional and genetic relations. Gene expression profiling of drought responsive genes in B. oleracea and Arabidopsis thaliana revealed significant differences in the gene expression pattern of a key regulator of ABA signalling—ABI1 PP2C. This finding prompted us to study genetic relations within the PP2Cs group A in the Brassica species. Twenty homologous B. oleracea sequences were identified and characterised as putative PP2C group A members. Phylogenetic analysis revealed that the B. oleracea homologues were closely related to the particular members of the A. thaliana PP2C. The genetic analysis corroborated the presence of two to three gene copies in B. oleracea in comparison to the nine unique PP2C genes in the A. thaliana genome. Gene expression analyses showed significant differences in PP2C gene expression pattern in B. oleracea. Our results indicate that PP2C‐based drought stress signalling in B. oleracea has evolved distinctly. Different reactions of particular B. oleracea PP2C genes to drought stress and ABA treatment indicate low conservation of gene expression patterns and functional divergence between B. oleracea and A. thaliana homologous genes.     

Functional innovations of three chronological mesohexaploid Brassica rapa genomes

Background

The Brassicaceae family is an exemplary model for studying plant polyploidy. The Brassicaceae knowledge-base includes the well-annotated Arabidopsis thaliana reference sequence; well-established evidence for three rounds of whole genome duplication (WGD); and the conservation of genomic structure, with 24 conserved genomic blocks (GBs). The recently released Brassica rapa draft genome provides an ideal opportunity to update our knowledge of the conserved genomic structures in Brassica, and to study evolutionary innovations of the mesohexaploid plant, B. rapa.

Results

Three chronological B. rapa genomes (recent, young, and old) were reconstructed with sequence divergences, revealing a trace of recursive WGD events. A total of 636 fast evolving genes were unevenly distributed throughout the recent and young genomes. The representative Gene Ontology (GO) terms for these genes were ‘stress response’ and ‘development’ both through a change in protein modification or signaling, rather than by enhancing signal recognition. In retention patterns analysis, 98% of B. rapa genes were retained as collinear gene pairs; 77% of those were singly-retained in recent or young genomes resulting from death of the ancestral copies, while others were multi-retained as long retention genes. GO enrichments indicated that single retention genes mainly function in the interpretation of genetic information, whereas, multi-retention genes were biased toward signal response, especially regarding development and defense. In the recent genome, 13,302, 5,790, and 20 gene pairs were multi-retained following Brassica whole genome triplication (WGT) events with 2, 3, and 4 homoeologous copies, respectively. Enriched GO-slim terms from B. rapa homomoelogues imply that a major effect of the B. rapa WGT may have been to acquire environmental adaptability or to change the course of development. These homoeologues seem to more frequently undergo subfunctionalization with spatial expression patterns compared with other possible events including nonfunctionalization and neofunctionalization.

Conclusion

We refined Brassicaceae GB information using the latest genomic resources, and distinguished three chronologically ordered B. rapa genomes. B. rapa genes were categorized into fast evolving, single- and multi-retention genes, and long retention genes by their substitution rates and retention patterns. Representative functions of the categorized genes were elucidated, providing better understanding of B. rapa evolution and the Brassica genus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-606) contains supplementary material, which is available to authorized users.     

Retention of protein complex membership by ancient duplicated gene products in budding yeast

To investigate functional divergence of gene duplicates, we examined the protein-protein interactions and coexistence in complexes of paralogs resulting from an ancient whole-genome duplication in yeast. Strikingly, half the surveyed paralog pairs were found to be co-clustered in protein complexes, and were more conserved and highly expressed than non-co-clustered paralogs; however, their discordant expression patterns and conservation rates indicate differential regulation of subfunctionalized paralogs. These results highlight the value of protein complex membership in studying functional divergence among gene duplicates.     

Molecular characterization of the flowering time gene FRIGIDA in Brassica genomes A and C

An important determinant of flowering time variation in Arabidopsis, the FRIGIDA (FRI) gene has not been until recently investigated in economically important Brassica species. In diploid Brassica species, this gene exists as two paralogous loci on chromosomes A3 and A4 (B. rapa; A genome), and C3 and C9 (B. oleracea; C genome). Each locus is represented by several genome-specific alleles, which are discerned primarily by polymorphisms in C- and especially N-terminal regions. Locus- and genome-specific sequences of two FRI paralogues are conserved almost completely in the subgenomes A and C of tetraploid B. napus. The phylogenetic analysis of available FRI sequences presumes that the duplication of FRI loci preceded speciation in the genus Brassica.     

The vernalization gene FRIGIDA in cultivated Brassica species

The repressor FLOWERING LOCUS C (FLC) holds a key position among the genes, which drive Arabidopsis floral transition along the vernalization pathway. The FRIGIDA (FRI) gene activates FLC expression, and the interplay of strong and weak alleles of FLC and FRI in many cases explains the variations in Arabidopsis requirement for cold induction. In annual and biennial life forms of Brassica, the variations in time to flower have been also related to FLC; whereas the place of FRI in the vernalization process has not been sufficiently elucidated. In contrast to Arabidopsis, FRI in Brassica genomes A and C and presumably B is represented by two expressible loci, FRI.a and FRI.b, each of them manifesting genome-specific polymorphisms. FRI.a and FRI.b sequences from diploid species B. rapa (genome A) and B. oleracea (genome C) are conserved (96–99% similarity) in subgenomes A and C of tetraploid species B. carinata (genome BC), B. juncea (genome AB), and B. napus (genome AC). Phylogenetic analysis of FRI sequences in the genus Brassica clearly discerns the lineages A/C and B, while in the family Brassicaceae, two FRI clusters discriminated by such analysis correspond to the lineages I (including the genus Arabidopsis) and II (including the genus Brassica). The origin of two FRI loci is discussed in the context of the Brassicaceae evolution via paleopolyploidy and subsequent genome reorganization.