Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies

Brassica rapa comprises several important cultivated vegetables and oil crops.Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous,thereby limiting extensive genetic and genomic analyses.Here,we report an improved assembly of the B.rapa genome(v3.0)using single-molecule sequencing,optical mapping,and chromosome conformation capture technologies(Hi-C).Relative to the previous reference genomes,our assembly features a contig N50 size of 1.45 Mb,representing a~30-fold improvement.We also identified a new event that occurred in the B.rapa genome~1.2 million years ago,when a long terminal repeat retrotransposon(LTR-RT)expanded.Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B.rapa genome.The B.rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B.rapa.This resource will facilitate breeding efforts in B.rapa,as well as comparative genomic analysis with other Brassica species.

A naturally occurring variation in the BrMAM-3 gene is associated with aliphatic glucosinolate accumulation in Brassica rapa leaves

Glucosinolate profiles significantly vary among Brassica rapa genotypes.However,the molecular basis of these variations is largely unknown.In this study,we investigated a major quantitative trait locus(QTL)controlling aliphatic glucosinolate accumulation in B.rapa leaves.The QTL,which encompasses three tandem MAM genes and two MYB genes,was detected in two BC2DH populations.Among the five-candidate genes,only the expression level of BrMAM-3(Bra013007)was significantly correlated with the accumulation of aliphatic glucosinolates in B.rapa leaves.We identified a naturally occurring insertion within exon 1 of BrMAM-3,which is predicted to be a loss-of-function mutation,as confirmed by qRT-PCR.We determined that the loss of function was associated with the low glucosinolate content in B.rapa accessions.Furthermore,overexpressing the BrMAM-3 gene resulted in an increase in total aliphatic glucosinolates in Arabidopsis transgenic lines.Our study provides insights into the molecular mechanism underlying the accumulation of aliphatic glucosinolates in B.rapa leaves,thereby facilitating in the manipulation of total aliphatic glucosinolate content in Brassica crops.

Development of InDel Markers for Brassica rapa Based on a High-resolution Melting Curve

Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-resolution melting(HRM)curve analysis.We designed primers for 252 InDels(≥3 bp)evenly distributed in the genome and tested gene polymorphisms with eight accessions.In total,208 markers were specifically amplified,and 148 InDels with polymorphism were genotyped successfully using HRM.We further analyzed the correlation with InDel size,GC number,and predicted the difference in Tm values(Tm)using 208 markers with specific amplification.We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-Tm,but not clearly correlated with the length of InDel.When the GC number within InDel was≥8,the successful rate exceeded 90.0%.When the predicted-Tm reached 0.5°C,the success rate was greater than 90.0%,and when it was≥0.6°C,the rate climbed to 100.0%,indicating their role as the optimal parameter for successful development of an applicable InDel marker.The polymorphic InDel markers can be easily genotyped using HRM.They are of great value in genetic analysis,construction of linkage map,and molecular marker-assisted selection in B.rapa.

Author Correction:Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies

Since the publication of this article,the authors have noticed that the total gene models(45,985),tandem arrays(2077),tandem genes(4963),redundancy removed(43,099),syntenic genes(39,858),nonsyntenic genes(3241),genes on chromosomes(45,411),genes on scaffolds(574)of B.rapa reference genome v3.0 were mistaken in the article.

Segmental Translocation Contributed to the Origin of the Brassica S-locus

Self-incompatibility(SI),which has recurred during the evolution of plants,is one of the most important cross-pollination mating systems.Three S-loci have been reported in Brassicaceae,namely,Arabidopsis lyrata(Al),Brassica(Br),and Leavenworthia alabamica(La)S-loci.Here,through multi-genomic comparative analysis of 20 species,we revealed that the most ancient S-locus was formed prior to the divergence of Brassicaceae lineage I and II.Itwas retained and inherited by Arabidopsis,as the Al S-locus in Brassicaceae lineage I.Furthermore,we found that the Br S-locus,which has been widely used in the breeding of Brassica crops to generate hybrid seeds,was formed through segmental translocation(ST)in the hexaploid ancestor of Brassica in Brassicaceae lineage II.The Br S-locus was evolved through a ST from one of the triplicated ancestral S-locus paralogs in the Brassica hexaploidy ancestor,while the other two S-locus paralogs were lost.Together with the previous discovery that the La S-locus was formed through a secondary origin in Brassicaceae lineage I,we conclude the monophyletic origin of Al and Br S-loci and clarify the evolutionary route of S-loci in the Brassicaceae family.Our findings will contribute to evolutionary studies and breeding applications of the S-locus in Brassicaceae.

Divergence of three BRX homoeologs in Brassica rapa and its effect on leaf morphology

The leafy head characteristic is a special phenotype of Chinese cabbage resulting from artificial selection during domestication and breeding.BREVIS RADIX(BRX)has been suggested to control root elongation,shoot growth,and tiller angle in Arabidopsis and rice.In Brassica rapa,three BrBRX homoeologs have been identified,but only BrBRX.1 and BrBRX.2 were found to be under selection in leaf-heading accessions,indicating their functional diversification in leafy head formation.Here,we show that these three BrBRX genes belong to a plant-specific BRX gene family but that they have significantly diverged from other BRX-like members on the basis of different phylogenetic classifications,motif compositions and expression patterns.Moreover,although the expression of these three BrBRX genes differed,compared with BrBRX.3,BrBRX.1,and BrBRX.2 displayed similar expression patterns.Arabidopsis mutant complementation studies showed that only BrBRX.1 could rescue the brx root phenotype,whereas BrBRX.2 and BrBRX.3 could not.However,overexpression of each of the three BrBRX genes in Arabidopsis resulted in similar pleiotropic leaf phenotypes,including epinastic leaf morphology,with an increase in leaf number and leaf petiole length and a reduction in leaf angle.These leaf traits are associated with leafy head formation.Further testing of a SNP(T/C)in BrBRX.2 confirmed that this allele in the heading accessions was strongly associated with the leaf-heading trait of B.rapa.Our results revealed that all three BrBRX genes may be involved in the leaf-heading trait,but they may have functionally diverged on the basis of their differential expression.

An optimized protocol for detecting guard cell specific gene expression by in situ RT-PCR in Brassica rapa

It is important to detect specific genes expressed in the guard cells,which control gas exchange and play key roles in response to drought and salt stresses.Due to the genetic transformation of Chinese cabbage(Brassica rapa)has not been well developed,in situ RT-PCR is a valuable option for detecting guard cell specific genes.We reported an optimized protocol of in situ RT-PCR by using an FAMA homologous gene Bra001929 in Brassica rapa.FAMA in Arabidopsis has been verified to be specially expressed in guard cells.We designed specific RT-PCR primers and optimized the protocol in terms of the(a)reverse transcription time,(b)blocking time,(c)antigen-antibody incubation time,and(d)washing temperature.Our approach provides a sensitive and effective in situ RT-PCR method for locating expression in the guard cells in Brassica rapa.Moreover,we proved the guard cell specific expression of Bra001929 in the epidermis indicating its’applicability as a marker gene for guard cells of Brassica rapa.

Identification and Analysis of Chemical Components in Zhideke Granules by UPLC-Q-Orbitrap HRMS

[Objectives]To identify and analyze chemical components in Zhideke Granules by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry(UPLC-Q-Orbitrap HRMS).[Methods]Zhideke Granules were isolated on a Thermo Hypersil Gold C18 column(2.1 mm×100 mm,1.9μm).The mobile phase was 0.1%formic acid acetonitrile and 0.1%formic acid aqueous solution(containing 10 mmol ammonium acetate)with gradient elution.Chemical components in Zhideke Granules were rapidly isolated and identified by HRMS in the positive and negative ion mode with full scan data-dependent two stage threshold-triggered mass modes(Full MS/dd-MS2).[Results]The secondary fragment ion information of the target compound was selected and compared with the compound reported in the databases and related literatures for further confirmation.In total,30 chemical compounds were identified,including 12 flavonoids and glycosides,9 organic acids,3 nitrogen-containing compounds,and 6 other components.[Conclusions]In this study,the UPLC-Q-Orbitrap HRMS was used for the first time to analyze the chemical components in Zhideke Granules.It is intended to provide a reference for the quality evaluation and further study of pharmacodynamic materials of Zhideke Granules.

Boron nitride nanofibers enhanced composite PEO-based solid-state polymer electrolytes for lithium metal batteries

Polyethylene oxide(PEO)-based solid-state polymer electrolytes(SPEs)are limited by their poor cyclic stability and inferior ionic conductivity for applicating in high-safety,long-cycling and high-energy-density lithium metal batteries.Herein,porous boron nitride nanofibers(BNNFs)are filled into PEO-based SPE,which significantly suppresses Li dendrites growth and enhances the electrochemical performance of Li metal battery.BNNFs with high porosity have more active sites to connect with PEO,which can effectively reduce the crystallinity of the PEO matrix and enhance its ionic conductivity.Moreover,owing to the hardness and good stability of BNNFs,BNNFs/PEO/Li TFSI electrolyte exhibits a wider electrochemical window,better mechanical property and higher thermal stability compared with PEO/Li TFSI electrolyte.Consequently,the Li symmetric cell composed of 1%BNNFs/PEO/Li TFSI performs good cyclic stability(>1800 h),and the Li||1%BNNFs/PEO/Li TFSI||LFP full battery shows obviously improved performances in charge-discharge polarization voltage,discharge specific capacity,rate performance and cyclic stability than the Li||PEO/Li TFSI||LFP battery.

Conserved noncoding sequences correlate with distant gene contacts in Arabidopsis and Brassica

Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput chromatin conformation capture(Hi-C)experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea.We then combined our results with published Hi-C data from Arabidopsis thaliana.We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene.Genes with higher numbers of conserved noncoding sequences(CNSs)nearby were more likely to have contact with distant genes.With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity.In addition,for the genes we studied,distant contacting genes with CNSs were more likely to be transcriptionally coordinated.These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors,correlating with distant contacts to ensure coordinated expression.This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.

Negatively charged insulated boron nitride nanofibers directing subsurface zinc deposition for dendrite-free zinc anodes

The practical application of aqueous zinc-ion batteries(ZIBs)is limited by the growth of dendrite during cycling.How to rationally design and construct an efficient artificial interface layer by selecting suitable building units to control the dendrite growth is still a challenge.Herein,a porous boron nitride nanofibers(BNNFs)artificial interface layer was constructed,and its working mechanisms were revealed by both experiments(electrochemical characterization and in-situ optical microscope)and theoretical calculations(density functional theory(DFT)and finite element simulation).The insulated BNNFs layer leads to position-selected electroplating between BNNFs layer and Zn foil.The unique negatively charged surface and porosity of BNNFs contribute to the self-concentrating and pumping features of Zn ions,thus suppressing the concentration polarization on the Zn surface.Additionally,densely arranged porous BNNFs have a shunt effect on Zn ions diffusion,resulting in uniform distributions of Zn ions and electric field.The introduced BNNFs layer not only makes Zn deposition uniform but also restrains the dendrite growth,therefore the Zn+BNNFs symmetric cells perform ultralong stable cycling for 1,600 h at 1 mA·cm–2 and more than 500 h at 10 mA·cm–2.Moreover,Zn+BNNFs||CNT/MnO2 battery presents a high initial capacity of 293.6 mAh·g–1 and an excellent retention rate of 97.6%at 1 A·g–1 after 400 cycles,while Zn||CNT/MnO2 battery only maintains 37.1%discharge capacity.This artificial interface layer with negatively charged BNNFs exhibits excellent dendrite-inhibit and may have enormous prospects in other metal batteries.

Variation in Plant Morphology and Sinigrin Content in Ethiopian Mustard(Brassica carinata L.)

Ethiopian mustard(Brassica carinata) is mainly grown as a leafy vegetable and oilseed crop. Sinigrin is predominant glucosinolate in the leaves of Ethiopian mustard. It is hydrolyzed by enzyme myrosinase to produce bioactive product specifically allyl-isothiocyanates. Due to sinigrin content, Ethiopian mustard has a mild flavor and it is a healthy addition to human diet. However, sinigrin concentration depends on genotype.Understanding variations of plant morphology and sinigrin content may be the first step for crop improvement. According to the analyses of various plant characteristics among ninety nine Ethiopian mustard accessions, we found significant differences in plant height, chlorophyll content, and petiole length. In addition, sinigrin content was determined using ultra-performance liquid chromatography tandem quadrupole mass spectrometers/tunable ultraviolet detector(UPLC-TQS/TUV). We found a significant difference in sinigrin content among leaves of 94 Ethiopian mustard accessions. Except for petiole length, the sinigrin content was negatively correlated with leaf area, leaf width, number of primary branches, and plant height. This information will help Ethiopian mustard breeders in screening breeding lines for new accessions.