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.     

Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus

Background and Aims

Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome A^rA^rC^cC^c) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation.

Methods

Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome AⁿAⁿCⁿCⁿ) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers.

Key Results

Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64·97 %) compared with D1-1 (32·23 %).

Conclusions

The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.     

Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

Background

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.

Results

A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.

Conclusions

Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.     

Quantitative trait loci for seed yield and yield-related traits, and their responses to reduced phosphorus supply in Brassica napus

Background and Aims

One of the key targets of breeding programmes in rapeseed (Brassica napus) is to develop high-yield varieties. However, the lack of available phosphorus (P) in soils seriously limits rapeseed production. The aim of this study was to dissect the genetic control of seed yield and yield-related traits in B. napus grown with contrasting P supplies.

Methods

Two-year field trials were conducted at one site with normal and low P treatments using a population of 124 recombinant inbred lines derived from a cross between ‘B104-2’ and ‘Eyou Changjia’. Seed yield, seed weight, seed number, pod number, plant height, branch number and P efficiency coefficient (PEC) were investigated. Quantitative trait locus (QTL) analysis was performed by composite interval mapping.

Key Results

The phenotypic values of most of the tested traits were reduced under the low P conditions. In total, 74 putative QTLs were identified, contributing 7·3–25·4 % of the phenotypic variation. Of these QTLs, 16 (21·6 %) were detected in two seasons and in the mean value of two seasons, and eight QTLs for two traits were conserved across P levels. Low-P-specific QTLs were clustered on chromosomes A1, A6 and A8. By comparative mapping between Arabidopsis and B. napus, 161 orthologues of 146 genes involved in Arabidopsis P homeostasis and/or yield-related trait control were associated with 45 QTLs corresponding to 23 chromosomal regions. Four gene-based markers developed from genes involved in Arabidopsis P homeostasis were mapped to QTL intervals.

Conclusions

Different genetic determinants were involved in controlling seed yield and yield-related traits in B. napus under normal and low P conditions. The QTLs detected under reduced P supply may provide useful information for improving the seed yield of B. napus in soils with low P availability in marker-assisted selection.     

Compositional studies and Biological activities of Perovskia abrotanoides Kar. oils

Background

Current study has been designed to evaluate the chemical composition of essential and fixed oils from stem and leaves of Perovskia abrotanoides and antioxidant and antimicrobial activities of these oils.

Results

GC-MS analysis of essential oil identified 19 compounds with (E)-9-dodecenal being the major component in stem and hexadecanoic acid in leaves. In contrast, GC-MS analysis of fixed oil showed 40 constituents with α-amyrin the major component in stem and α-copaene in leaves. The antioxidant activity showed the highest value of 76.7% in essential oil from leaves in comparison with fixed oil from stem (45.9%) through inhibition of peroxidation in linoleic acid system. The antimicrobial assay tested on different microorganisms (e.g. E. coli, S. aureus, B. cereus, Nitrospira, S. epidermis, A. niger, A. flavus and C. albicans) showed the higher inhibition zone at essential oil from leaves (15.2 mm on B. cereus) as compared to fixed oil from stem (8.34 mm on S. aureus) and leaves (11.2 mm on S. aureus).

Conclusions

The present study revealed the fact that essential oil analyzed from Perovskia abrotanoides stem and leaves could be a promising source of natural products with potential antioxidant and antimicrobial activities, as compared to fixed oil.     

A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus

Background

Citrus represents a crop of global importance both in economic impact and significance to nutrition. Citrus production worldwide is threatened by the disease Huanglongbing (HLB), caused by the phloem-limited pathogen Candidatus Liberibacter spp.. As a source of stable HLB-resistance has yet to be identified, there is considerable interest in characterization of novel disease-associated citrus genes.

Results

A gene family of Small Cyclic Amphipathic Peptides (SCAmpPs) in citrus is described. The citrus genomes contain 100–150 SCAmpPs genes, approximately 50 of which are represented in the citrus EST database. These genes encode small ~50 residue precursor proteins that are post-translationally processed, releasing 5–10 residue cyclic peptides. The structures of the SCAmpPs genes are highly conserved, with the small coding domains interrupted by a single intron and relatively extended untranslated regions. Some family members are very highly transcribed in specific citrus tissues, as determined by representation in tissue-specific cDNA libraries. Comparison of the ESTs of related SCAmpPs revealed an unexpected evolutionary profile, consistent with targeted mutagenesis of the predicted cyclic peptide domain. The SCAmpPs genes are displayed in clusters on the citrus chromosomes, with apparent association with receptor leucine-rich repeat protein arrays. This study focused on three SCAmpPs family members with high constitutive expression in citrus phloem. Unexpectedly high sequence conservation was observed in the promoter region of two phloem-expressed SCAmpPs that encode very distinct predicted cyclic products. The processed cyclic product of one of these phloem SCAmpPs was characterized by LC-MS-MS analysis of phloem tissue, revealing properties consistent with a K⁺ ionophore.

Conclusions

The SCAmpPs amino acid composition, protein structure, expression patterns, evolutionary profile and chromosomal distribution are consistent with designation as ribosomally synthesized defense-related peptides.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1486-4) contains supplementary material, which is available to authorized users.     

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

Background and Aims

Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5.

Methods

A large F₂ population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5.

Key Results

BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence.

Conclusions

This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus.     

A bi-filtering method for processing single nucleotide polymorphism array data improves the quality of genetic map and accuracy of quantitative trait locus mapping in doubled haploid populations of polyploid Brassica napus

Background

Single nucleotide polymorphism (SNP) markers have a wide range of applications in crop genetics and genomics. Due to their polyploidy nature, many important crops, such as wheat, cotton and rapeseed contain a large amount of repeat and homoeologous sequences in their genomes, which imposes a huge challenge in high-throughput genotyping with sequencing and/or array technologies. Allotetraploid Brassica napus (AACC, 2n = 4x = 38) comprises of two highly homoeologous sub-genomes derived from its progenitor species B. rapa (AA, 2n = 2x = 20) and B. oleracea (CC, 2n = 2x = 18), and is an ideal species to exploit methods for reducing the interference of extensive inter-homoeologue polymorphisms (mHemi-SNPs and Pseudo-simple SNPs) between closely related sub-genomes.

Results

Based on a recent B. napus 6K SNP array, we developed a bi-filtering procedure to identify unauthentic lines in a DH population, and mHemi-SNPs and Pseudo-simple SNPs in an array data matrix. The procedure utilized both monomorphic and polymorphic SNPs in the DH population and could effectively distinguish the mHemi-SNPs and Pseudo-simple SNPs that resulted from superposition of the signals from multiple SNPs. Compared with conventional procedure for array data processing, the bi-filtering method could minimize the pseudo linkage relationship caused by the mHemi-SNPs and Pseudo-simple SNPs, thus improving the quality of SNP genetic map. Furthermore, the improved genetic map could increase the accuracies of mapping of QTLs as demonstrated by the ability to eliminate non-real QTLs in the mapping population.

Conclusions

The bi-filtering analysis of the SNP array data represents a novel approach to effectively assigning the multi-loci SNP genotypes in polyploid B. napus and may find wide applications to SNP analyses in polyploid crops.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1559-4) contains supplementary material, which is available to authorized users.     

A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

Background

To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.

Results

Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.

Conclusions

The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.