Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis,phylogenetic analysis,and expression profiling

Background

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa.

Results

We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.

Conclusions

This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1655-5) contains supplementary material, which is available to authorized users.     

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

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Beyond genomic variation - comparison and functional annotation of three Brassica rapa genomes: a turnip,a rapid cycling and a Chinese cabbage

Background

Brassica rapa is an economically important crop species. During its long breeding history, a large number of morphotypes have been generated, including leafy vegetables such as Chinese cabbage and pakchoi, turnip tuber crops and oil crops.

Results

To investigate the genetic variation underlying this morphological variation, we re-sequenced, assembled and annotated the genomes of two B. rapa subspecies, turnip crops (turnip) and a rapid cycling. We then analysed the two resulting genomes together with the Chinese cabbage Chiifu reference genome to obtain an impression of the B. rapa pan-genome. The number of genes with protein-coding changes between the three genotypes was lower than that among different accessions of Arabidopsis thaliana, which can be explained by the smaller effective population size of B. rapa due to its domestication. Based on orthology to a number of non-brassica species, we estimated the date of divergence among the three B. rapa morphotypes at approximately 250,000 YA, far predating Brassica domestication (5,000-10,000 YA).

Conclusions

By analysing genes unique to turnip we found evidence for copy number differences in peroxidases, pointing to a role for the phenylpropanoid biosynthesis pathway in the generation of morphological variation. The estimated date of divergence among three B. rapa morphotypes implies that prior to domestication there was already considerably divergence among B. rapa genotypes. Our study thus provides two new B. rapa reference genomes, delivers a set of computer tools to analyse the resulting pan-genome and uses these to shed light on genetic drivers behind the rich morphological variation found in B. rapa.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-250) contains supplementary material, which is available to authorized users.     

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana

Background

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.

Results

Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.

Conclusion

This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-3) contains supplementary material, which is available to authorized users.     

Genome-wide identification and Phylogenic analysis of kelch motif containing ACBP in Brassica napus

Background

Acyl-coA binding proteins (ACBPs) bind long chain acyl-CoA esters with very high affinity. Their possible involvement in fatty acid transportation from the plastid to the endoplasmic reticulum, prior to the formation of triacylglycerol has been suggested. Four classes of ACBPs were identified in Arabidopsis thaliana: the small ACBPs, the large ACBPs, the ankyrin repeats containing ACBPs and the kelch motif containing ACBPs. They differed in structure and in size, and showed multiple important functions. In the present study, Brassica napus ACBPs were identified and characterized.

Results

Eight copies of kelch motif ACBPs were cloned, it showed that B. napus ACBPs shared high amino acid sequence identity with A. thaliana, Brassica rapa and Brassica oleracea. Furthermore, phylogeny based on domain structure and comparison map showed the relationship and the evolution of ACBPs within Brassicaceae family: ACBPs evolved into four separate classes with different structure. Chromosome locations comparison showed conserved syntenic blocks.

Conclusions

ACBPs were highly conserved in Brassicaceae. They evolved from a common ancestor, but domain duplication and rearrangement might separate them into four distinct classes, with different structure and functions. Otherwise, B. napus inherited kelch motif ACBPs from ancestor conserving chromosomal location, emphasizing preserved synteny block region. This study provided a first insight for exploring ACBPs in B. napus, which supplies a valuable tool for crop improvement in agriculture.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1735-6) contains supplementary material, which is available to authorized users.     

Distribution of calcium (Ca) and magnesium (Mg) in the leaves of Brassica rapa under varying exogenous Ca and Mg supply

Background and Aims

Leafy vegetable Brassica crops are an important source of dietary calcium (Ca) and magnesium (Mg) and represent potential targets for increasing leaf Ca and Mg concentrations through agronomy or breeding. Although the internal distribution of Ca and Mg within leaves affects the accumulation of these elements, such data are not available for Brassica. The aim of this study was to characterize the internal distribution of Ca and Mg in the leaves of a vegetable Brassica and to determine the effects of altered exogenous Ca and Mg supply on this distribution.

Methods

Brassica rapa ssp. trilocularis ‘R-o-18’ was grown at four different Ca:Mg treatments for 21 d in a controlled environment. Concentrations of Ca and Mg were determined in fully expanded leaves using inductively coupled plasma-mass spectrometry (ICP-MS). Internal distributions of Ca and Mg were determined in transverse leaf sections at the base and apex of leaves using energy-dispersive X-ray spectroscopy (EDS) with cryo-scanning electron microscopy (cryo-SEM).

Key Results

Leaf Ca and Mg concentrations were greatest in palisade and spongy mesophyll cells, respectively, although this was dependent on exogenous supply. Calcium accumulation in palisade mesophyll cells was enhanced slightly under high Mg supply; in contrast, Mg accumulation in spongy mesophyll cells was not affected by Ca supply.

Conclusions

The results are consistent with Arabidopsis thaliana and other Brassicaceae, providing phenotypic evidence that conserved mechanisms regulate leaf Ca and Mg distribution at a cellular scale. The future study of Arabidopsis gene orthologues in mutants of this reference B. rapa genotype will improve our understanding of Ca and Mg homeostasis in plants and may provide a model-to-crop translation pathway for targeted breeding.     

Comparative genomics of the Bifidobacterium breve taxon

Background

Bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, where their presence is positively correlated with the host’s health status. In this study, we assessed the genomes of thirteen representatives of Bifidobacterium breve, which is not only a frequently encountered component of the (adult and infant) human gut microbiota, but can also be isolated from human milk and vagina.

Results

In silico analysis of genome sequences from thirteen B. breve strains isolated from different environments (infant and adult faeces, human milk, human vagina) shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. These latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign DNA (i.e. CRISPR loci and restriction/modification systems), and genes that encode enzymes responsible for carbohydrate fermentation. Gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol.

Conclusions

Genome analysis of thirteen representatives of the B. breve species revealed that the deduced pan-genome exhibits an essentially close trend. For this reason our analyses suggest that this number of B. breve representatives is sufficient to fully describe the pan-genome of this species. Comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of B. breve.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-170) contains supplementary material, which is available to authorized users.     

Diversity of the cell-wall associated genomic island of the archaeon Haloquadratum walsbyi

Background

Haloquadratum walsbyi represents up to 80 % of cells in NaCl-saturated brines worldwide, but is notoriously difficult to maintain under laboratory conditions. In order to establish the extent of genetic diversity in a natural population of this microbe, we screened a H. walsbyi enriched metagenomic fosmid library and recovered seven novel version of its cell-wall associated genomic island. The fosmid inserts were sequenced and analysed.

Results

The novel cell-wall associated islands delineated two major clades within H. walsbyi. The islands predominantly contained genes putatively involved in biosynthesis of surface layer, genes encoding cell surface glycoproteins and genes involved in envelope formation. We further found that these genes are maintained in the population and that the diversity of this region arises through homologous recombination but also through the action of mobile genetic elements, including viruses.

Conclusions

The population of H. walsbyi in the studied saltern brine is composed of numerous clonal lineages that differ in surface structures including the cell wall. This type of variation probably reflects a number of mechanisms that minimize the infection rate of predating viruses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1794-8) contains supplementary material, which is available to authorized users.     

Seed colour loci, homoeology and linkage groups of the C genome chromosomes revealed in Brassica rapa-B. oleracea monosomic alien addition lines

Background and Aims

Brassica rapa and B. oleracea are the progenitors of oilseed rape B. napus. The addition of each chromosome of B. oleracea to the chromosome complement of B. rapa results in a series of monosomic alien addition lines (MAALs). Analysis of MAALs determines which B. oleracea chromosomes carry genes controlling specific phenotypic traits, such as seed colour. Yellow-seeded oilseed rape is a desirable breeding goal both for food and livestock feed end-uses that relate to oil, protein and fibre contents. The aims of this study included developing a missing MAAL to complement an available series, for studies on seed colour control, chromosome homoeology and assignment of linkage groups to B. oleracea chromosomes.

Methods

A new batch of B. rapa–B. oleracea aneuploids was produced to generate the missing MAAL. Seed colour and other plant morphological features relevant to differentiation of MAALs were recorded. For chromosome characterization, Snow''s carmine, fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used.

Key Results

The final MAAL was developed. Morphological traits that differentiated the MAALs comprised cotyledon number, leaf morphology, flower colour and seed colour. Seed colour was controlled by major genes on two B. oleracea chromosomes and minor genes on five other chromosomes of this species. Homoeologous pairing was largely between chromosomes with similar centromeric positions. FISH, GISH and a parallel microsatellite marker analysis defined the chromosomes in terms of their linkage groups.

Conclusions

A complete set of MAALs is now available for genetic, genomic, evolutionary and breeding perspectives. Defining chromosomes that carry specific genes, physical localization of DNA markers and access to established genetic linkage maps contribute to the integration of these approaches, manifested in the confirmed correspondence of linkage groups with specific chromosomes. Applications include marker-assisted selection and breeding for yellow seeds.