Comparative Transcriptome Analysis Points to the Biological Processes of Hybrid Incompatibility between Brassica napus and B. oleracea.

Improving Brassica napus via introgression of the genome components from its parental species, B. oleracea and B. rapa, is an important breeding strategy. Interspecific hybridization between B. napus and B. rapa is compatible with high rate of survival ovules, while the hybridization between B. napus and B. oleracea is incompatible with the high occurrence of embryo abortion. To understand the diverse embryo fate in the two interspecific hybridizations, here, the siliques of B. napus pollinated with B. oleracea (AE) and B. rapa (NE) were employed for transcriptome sequencing at 8 and 16 days after pollination. Compared to NE and the parental line of B. napus, more specific differentially expressed genes (DEGs) (1274 and 1698) were obtained in AE and the parental line of B. napus at 8 and 16 days after pollination (DAP). These numbers were 51 and 5.8 times higher than the number of specific DEGs in NE and parental line of B. napus at 8 and 16 DAP, respectively, suggesting more complex transcriptional changes in AE. Most of DEGs in the terms of cell growth and cell wall formation exhibited down-regulated expression patterns (96(down)/131(all) in AE8, 174(down)/235(all) in AE16), while most of DEGs in the processes of photosynthesis, photorespiration, peroxisome, oxidative stress, and systemic acquired resistance exhibited up-regulated expression patterns (222(up)/304(all) in AE8, 214(up)/287(all) in AE16). This is in accordance with a high level of reactive oxygen species (ROS) in the siliques of B. napus pollinated with B. oleracea. Our data suggest that the disorder of plant hormone metabolism, retardation of cell morphogenesis, and the accumulation of ROS may be associated with hybrid incompatibility between B. napus and B. oleracea.

A 215-bp indel at intron I of BoFLC2 affects flowering time in Brassica oleracea var. capitata during vernalization.

KEY MESSAGE: In response to cold, a 215-bp deletion at intron I of BoFLC2 slows its silencing activity by feedback to the core genes of the PHD-PRC2 complex, resulting in late flowering in cabbage. Cabbage is a plant-vernalization-responsive flowering type. In response to cold, BoFLC2 is an important transcription factor, which allows cabbage plants to remain in the vegetative phase. However, there have been few reports on the detailed and functional effects of genetic variation in BoFLC2 on flowering time in cabbage. Herein, BoFLC2E and BoFLC2L, cloned from extremely early and extremely late flowering cabbages, respectively, exhibited a 215-bp indel at intron I, three non-synonymous SNPs and a 3-bp indel at exon II. BoFLC2L was found to be related to late flowering, as verified in 40 extremely early/late flowering accessions, a diverse set of cabbage inbred lines and two F2 generations by using indel-FLC2 marker. Among the genetic variation of BoFLC2, the 215-bp deletion at intron I was the main reason for the delayed flowering time, as verified in the transgenic progenies of seed-vernalization-responsive Arabidopsis thaliana (Col) and rapid cycler B. oleracea (TO1000, boflc2). This is the first report to show that the intron I indel of BoFLC2 affects the flowering time of cabbage. Although the intron I 215-bp indel between BoFLC2E and BoFLC2L did not cause alternative splicing, it slowed BoFLC2L silencing during vernalization and feedback to the core genes of the PHD-PRC2 complex, resulting in their lower transcription levels. Our study not only provides an effective molecular marker-assisted selective strategy for identifying bolting-resistant resources and breeding improved varieties in cabbage, but also provides an entry point for exploring the mechanisms of flowering time in plant-vernalization-responsive plants.

Dexmedetomidine attenuates acute stress-induced liver injury in rats by regulating the miR-34a-5p/ROS/JNK/p38 signaling pathway.

Dexmedetomidine (DEX) protects against acute stress-induced liver injury, but what's less clear lies in the specific mechanism. To elucidate the specific mechanism underlying DEX on acute stress-induced liver injury, an in vivo model was constructed on rats with acute stress-induced liver injury by 15 min of exhaustive swimming and 3 hr of immobilization. DEX (30 µg/kg) or miR-34a-5p agomir was injected into model rats. Open field test was used to verify the establishment of the model. Liver injury was observed by hematoxylin-eosin (H&E) staining. Contents of norepinephrine (NE), alanine aminotransfease (ALT) and aspartate aminotransferase (AST) in serum of rats were detected by enzyme-linked immunosorbent assay (ELISA) and those of oxidative stress markers (reactive oxygen species (ROS), Malondialdehyde (MDA), Glutathione (GSH), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX)) were measured using commercial kits. Apoptosis of hepatocytes was detected by Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Western blot was performed to detect the expressions of SOD2, COX-2, cytochrome C, Cleaved caspase 3, Bax, Bcl-2, P-JNK, JNK, P-p38, p38 and c-AMP, p-PKA and PKA in liver tissues. As a result, liver injury in model rat was alleviated by DEX. DEX attenuated the increase in the levels of NE, ALT, AST, MDA, ROS, apoptosis, SOD2, COX-2, Cytochrome C, cleaved caspase 3, Bax, and P-JNK, P-p38, c-AMP, P-PKA and miR-34a-5p, and the decrease in the levels of SOD, GPX, GSH and Bcl-2 in model rats. Furthermore, miR-34a-5p overexpression could partly reverse the effects of DEX. Collectively, DEX could alleviate acute stress-induced liver injury through ROS/JNK/p38 signaling pathway via downregulation of miR-34a-5p.

A New Probabilistic Ellipse Imaging Method Based on Adaptive Signal Truncation for Ultrasonic Guided Wave Defect Localization on Pressure Vessels.

Pressure vessels are prone to defects due to environmental conditions, which may cause serious safety hazards to industrial production. The probabilistic ellipse imaging method, based on ultrasonic guided wave, is a common method for locating defects on plate-like structures. In this paper, the research showed that the accuracy of the traditional probabilistic ellipse imaging method was severely affected by the truncation length of the signal. In order to improve the defect location accuracy of the probabilistic elliptic imaging algorithm, an adaptive signal truncation method based on signal difference analysis was proposed, and a novel probabilistic elliptic imaging method was developed. Firstly, the relationship model between the signal difference coefficient (SDC) and the distance coefficient was constructed. Through this model, the distance coefficient of each group signal can be calculated, so that the adaptive truncation length for each group of signals can be determined and the truncated signals used for defect imaging. Secondly, in order to improve the robustness of the new imaging method, the relationship between the defect location accuracy and SDC thresholds were investigated and the optimal threshold was determined. The experimental results showed that the probabilistic ellipse imaging algorithm, based on the new adaptive signal truncation method, can effectively locate a single defect on a pressure vessel.

Identification of Candidate Genes for Clubroot-Resistance in Brassica oleracea Using Quantitative Trait Loci-Sequencing.

Clubroot caused by Plasmodiophora brassicae is a devastating disease of cabbage (Brassica oleracea). To identify quantitative trait loci (QTLs) for clubroot resistance (CR) in B. oleracea, genomic resequencing was carried out in two sets of extreme pools, group I and group II, which were constructed separately from 110 and 74 F2 cloned lines derived from the cross between clubroot-resistant (R) cabbage "GZ87" (against race 4) and susceptible (S) cabbage "263." Based on the QTL-sequencing (QTL-Seq) analysis of group I and group II, three QTLs (i.e., qCRc7-2, qCRc7-3, and qCRc7-4) were determined on the C07 chromosome. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation, and two potential candidate genes (i.e., Bol037115 and Bol042270), which exhibiting upregulation after inoculation in the R pool but downregulation in the S pool, were identified from the three QTLs on C07. A functional marker "SWU-OA" was developed from qCRc7-4 on C07, exhibiting ∼95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on resistance genes against P. brassicae and may accelerate the breeding process of B. oleracea with CR.

Development of Ogura CMS restorers in Brassica oleracea subspecies via direct RfoB gene transformation.

KEY MESSAGE: The Ogura CMS RfoB restorer developing via RfoB gene transformation was utilized to produce specific morphological Ogura CMS restorers and clubroot resistance lines in Brassica oleracea subspecies. Brassica oleracea vegetables including cabbage, cauliflower, kohlrabi, Brussels sprouts and Chinese kale are morphologically very different despite being members of the same species. The Ogura cytoplasmic male sterility (CMS) system is the most stable strategy for the hybrid breeding of these species. However, this limits the utilization of some excellent genes due to the lack of fertile restorer genes in the system. Herein, to efficaciously use Ogura CMS, the Ogura CMS RfoB restorer was produced by transforming the modified RfoB restorer gene into the Ogura CMS line 'CMS2016' of B. oleracea var. capitata. This gene was shown to recover fertility of natural Ogura CMS lines in B. oleracea subspecies and create transient Ogura CMS RfoB restorers such as the clubroot resistance Ogura CMS RfoB restorer. Interestingly, clubroot resistant individuals without transgenic elements were screened in the progenies of hybrids between B. oleracea inbred lines and the clubroot resistance Ogura CMS RfoB restorer. In addition, 18 different morphological Ogura CMS restorers were developed to specifically recover fertile of Ogura CMS cultivars in B. oleracea subspecies.

Fabrication of a Sensitive Strain and Pressure Sensor from Gold Nanoparticle-Assembled 3D-Interconnected Graphene Microchannel-Embedded PDMS.

Manufacture of uniform, sensitive, and durable microtextured sensing materials is one of the greatest challenges for pressure sensors and electronic skins. Reported in this article is a gold nanoparticle-assembled, 3D-interconnected, graphene microchannel-embedded PDMS (3D GMC-PDMS) film for strain and pressure sensors. The film consists of porous nickel foam with its inner walls coated by multilayer graphene. Embedding in PDMS with etching removal of the Ni yields a 3D GMC-PDMS. Coating the inner walls with Au nanoparticles yields an Au nanoparticle-assembled 3D GMC-PDMS (AuNPs-GMC-PDMS) film, which is useful as an ultrasensitive pressure and strain sensor. This sensor exhibits a wide detection range (∼50 kPa) and ultrahigh sensitivity of 5.37, 1.56, and 0.5 kPa-1 in the ranges of <1, 1-10, and 10-50 kPa, respectively. Its lower detection limit is 4.4 Pa, its response time is 20 ms, and its strain factor is up to 15. Comparison of a AuNPs-GMC-PDMS film with a 3D GMC-PDMS film reveals a sensitivity improvement of 40 times in the 0-1 kPa pressure range and a gauge factor of more than 4 times in the 0-30% tensile strain range. The device has broad applications as a traditional or wearable medical sensor.

Effect of Phenolic Particles on Mechanical and Thermal Conductivity of Foamed Sulphoaluminate Cement-Based Materials.

Foamed concrete materials based on sulpoaluminate cement were prepared by the chemical foaming method. The effects of water-cement ratio, foaming agent, and foaming stabilizer on the mechanical and thermal properties of foamed concrete were studied. Meanwhile, a portion of cement was replaced with foamed phenolic particles to further optimize the performance of foamed concrete; the results show that when the water-cement ratio was 0.53, the foaming agent content was 5%, the foam stabilizer was 1%, and the substitution of phenolic particles was 20%, the performance indexes of foamed concrete were the best. Methods, describing briefly the main methods or treatments applied: dry density was 278.4 kg/m3, water absorption was 19.9%, compressive strength was 3.01 MPa, and thermal conductivity was 0.072 W/(m·K). By the pore structure analysis of the foamed concrete suing Micro-CT, it was found that when the replacement amount of phenolic particles was 20%, the pore size of foamed concrete was relatively uniform, the minimum D90 was 225 µm respectively. The combination of organic and inorganic matrix and optimized pore structure improved the performance of foamed concrete.

CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system.

Cabbage (Brassica oleracea var. capitata) is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering. These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens. In this study, we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage. Using the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK, and the male-sterility-associated gene BoMS1 as the target genes, multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs. The BoSRK3 gene mutation suppressed self-incompatibility completely, converting the self-incompatible line into a self-compatible line. In addition, the BoMS1 gene mutation produced a completely male-sterile mutant, which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation, enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination. Interestingly, higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter. In addition, mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the "seed" region in the spacer sequence compared with the target sgRNAs. Collectively, our results demonstrate that the CRISPR/Cas9 system, coupled with an endogenous tRNA-processing system, is an efficient tool to improve cabbage traits.

Genetic characterization and fine mapping for multi-inflorescence in Brassica napus L.

KEY MESSAGE: A major QTL for multi-inflorescence was mapped to a 27.18-kb region on A05 in Brassica napus by integrating QTL mapping, microarray analysis and whole-genome sequencing. Multi-inflorescence is a desirable trait for the genetic improvement of rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait is not well understood. In the present study, a doubled haploid (DH) population derived from a cross between single- and multi-inflorescence lines was investigated for the penetrance of multi-inflorescence across 3 years and genotyped with 257 simple sequence repeat and sequence-related amplified polymorphism loci. A major quantitative trait locus (QTL) for penetrance of multi-inflorescence was mapped to a 9.31-Mb region on chromosome A05, explaining 45.81% of phenotypic variance on average. Subsequently, 13 single-inflorescence and 15 multi-inflorescence DH lines were genotyped with the Brassica microarray, and the QTL interval of multi-inflorescence was narrowed to a 0.74-Mb region with 37 successive single nucleotide polymorphisms between single- and multi-inflorescence groups. A 27.18-kb QTL interval was detected by screening 420 recessive F2 individuals with genome-specific markers. These results will be valuable for gene cloning and molecular breeding of multi-inflorescence in rapeseed.

Identification of Quantitative Trait Loci for Clubroot Resistance in Brassica oleracea With the Use of Brassica SNP Microarray.

Increasing clubroot resistance (CR) of Brassica oleracea by ascertaining the molecular mechanisms has been the key focus in modern B. oleracea breeding. In order to identify the quantitative trait loci (QTLs) associated with CR in B. oleracea, 94 F2 vegetative lines which were developed by tissue culture of selfed seeds from the F1 generation between a clubroot-resistant B. oleracea inbred line and a susceptible line, were identified for disease incidence and six CR-associated traits under a lab inoculation by Plasmodiophora brassicae and were genotyped with the 60K Brassica SNP array. Significant correlations were detected for numbers of fibrous roots and P. brassicae content in roots with disease incidence. Nine linkage groups were constructed from 565 bins which covered around 3,000 SNPs, spanning 1,028 cM of the B. oleracea genome with an average distance of 1.82 cM between adjacent bins. A total of 23 QTLs were identified for disease incidence and the other two correlated traits, individually explaining 6.1-17.8% of the phenotypic variation. Several overlaps were detected among traits, including one three-traits-overlapped locus on linkage group C08 and two important overlapped regions between the two CR-associated traits on C06. The QTLs were compared with known CR loci/genes and the novelty of our QTLs was discussed.

Microspore culture reveals high fitness of B. napus-like gametes in an interspecific hybrid between Brassica napus and B. oleracea.

The strategies of crossing B. napus with parental species play important role in broadening and improving the genetic basis of B. napus by the introgression of genetic resources from parental species. With these strategies, it is easy to select new types of B. napus, but difficult to select new types of B. rapa or B. oleracea by self-pollination. This characteristic may be a consequence of high competition with B. napus gametes. To verify the role of gamete viability in producing new B. napus individuals, the meiotic chromosome behavior of the interspecific hybrid between B. napus (Zhongshuang 9) and B. oleracea (6m08) was studied, and microspore-derived (MD) individuals were analyzed. The highest fitness of the 9:19 (1.10%) pattern was observed with a 5.49-fold higher than theoretical expectation among the six chromosome segregation patterns in the hybrid. A total of 43 MD lines with more than 14 chromosomes were developed from the hybrid, and 8 (18.6%) of them were B. napus-like (n = 19) type gametes, having the potential to broaden the genetic basis of natural B. napus (GD = 0.43 ± 0.04). It is easy to produce B. napus-like gametes with 19 chromosomes, and these gametes showed high fitness and competition in the microspore-derived lines, suggesting it might be easy to select new types of B. napus from the interspecific hybrid between B. napus and B. oleracea.

Identification of Polymorphisms Associated with Drought Adaptation QTL in Brassica napus by Resequencing.

Brassica napus is a globally important oilseed for which little is known about the genetics of drought adaptation. We previously mapped twelve quantitative trait loci (QTL) underlying drought-related traits in a biparental mapping population created from a cross between winter and spring B. napus cultivars. Here we resequence the genomes of the mapping population parents to identify genetic diversity across the genome and within QTL regions. We sequenced each parental cultivar on the Illumina HiSeq platform to a minimum depth of 23 × and performed a reference based assembly in order to describe the molecular variation differentiating them at the scale of the genome, QTL and gene. Genome-wide patterns of variation were characterized by an overall higher single nucleotide polymorphism (SNP) density in the A genome and a higher ratio of nonsynonymous to synonymous substitutions in the C genome. Nonsynonymous substitutions were used to categorize gene ontology terms differentiating the parent genomes along with a list of putative functional variants contained within each QTL. Marker assays were developed for several of the discovered polymorphisms within a pleiotropic QTL on chromosome A10. QTL analysis with the new, denser map showed the most associated marker to be that developed from an insertion/deletion polymorphism located in the candidate gene Bna.FLC.A10, and it was the only candidate within the QTL interval with observed polymorphism. Together, these results provide a glimpse of genome-wide variation differentiating annual and biennial B. napus ecotypes as well as a better understanding of the genetic basis of root and drought phenotypes.

Transfer of sclerotinia resistance from wild relative of Brassica oleracea into Brassica napus using a hexaploidy step.

KEY MESSAGE: Sclerotinia resistance was transferred into rapeseed from a wild relative of Brassica oleracea (B. incana) using hexaploids derived from crosses between B. incana and rapeseed as a bridge. A high level of resistance against Sclerotinia sclerotiorum has been documented in wild Brassica oleracea, but not in cultivated rapeseed (Brassica napus). To transfer sclerotinia resistance from a wild relative into rapeseed, a strategy was proposed using hexaploids (AACCCC) derived from crosses between the wild B. oleracea-related B. incana genotype 'C01' and the Chinese rapeseed variety 'Zhongshuang 9' as a bridge. Progenies (BC1F1) generated by backcrossing the hexaploid to 'Zhongshuang 9' could be generated with a high crossability (average 18.3 seeds per pod). Seventy-three individuals in BC1F1 were firstly screened for resistance with five molecular markers linked to the major resistance QTL on chromosome C09 in 'C01', and 11 individuals harboring resistance loci were selected to develop vegetative clones. Of these, five exhibited significantly higher resistance than 'Zhongshuang 9' and the most resistant individual was chosen to develop the BC1F2 progeny. Finally, five individual genotypes with nearly twofold higher resistance than 'Zhongshuang 9' were found among 100 BC1F2 individuals by using marker-assisted selection and resistance evaluation. Hereof, one rapeseed-type individual with 38 chromosomes and good self-fertility (15.0 ± 3.56 seeds/pod) was identified. Our results indicate that the proposed strategy is effective for transferring sclerotinia resistance from a wild relative of B. oleracea into rapeseed.

Mechanical resilience and cementitious processes in Imperial Roman architectural mortar.

The pyroclastic aggregate concrete of Trajan's Markets (110 CE), now Museo Fori Imperiali in Rome, has absorbed energy from seismic ground shaking and long-term foundation settlement for nearly two millenia while remaining largely intact at the structural scale. The scientific basis of this exceptional service record is explored through computed tomography of fracture surfaces and synchroton X-ray microdiffraction analyses of a reproduction of the standardized hydrated lime-volcanic ash mortar that binds decimeter-sized tuff and brick aggregate in the conglomeratic concrete. The mortar reproduction gains fracture toughness over 180 d through progressive coalescence of calcium-aluminum-silicate-hydrate (C-A-S-H) cementing binder with Ca/(Si+Al) ≈ 0.8-0.9 and crystallization of strätlingite and siliceous hydrogarnet (katoite) at ≥ 90 d, after pozzolanic consumption of hydrated lime was complete. Platey strätlingite crystals toughen interfacial zones along scoria perimeters and impede macroscale propagation of crack segments. In the 1,900-y-old mortar, C-A-S-H has low Ca/(Si+Al) ≈ 0.45-0.75. Dense clusters of 2- to 30-µm strätlingite plates further reinforce interfacial zones, the weakest link of modern cement-based concrete, and the cementitious matrix. These crystals formed during long-term autogeneous reaction of dissolved calcite from lime and the alkali-rich scoriae groundmass, clay mineral (halloysite), and zeolite (phillipsite and chabazite) surface textures from the Pozzolane Rosse pyroclastic flow, erupted from the nearby Alban Hills volcano. The clast-supported conglomeratic fabric of the concrete presents further resistance to fracture propagation at the structural scale.

DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5'-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5'-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

A large-scale introgression of genomic components of Brassica rapa into B. napus by the bridge of hexaploid derived from hybridization between B. napus and B. oleracea.

Brassica rapa (AA) has been used to widen the genetic basis of B. napus (AACC), which is a new but important oilseed crop worldwide. In the present study, we have proposed a strategy to develop new type B. napus carrying genomic components of B. rapa by crossing B. rapa with hexaploid (AACCCC) derived from B. napus and B. oleracea (CC). The hexaploid exhibited large flowers and high frequency of normal chromosome segregation, resulting in good seed set (average of 4.48 and 12.53 seeds per pod by self and open pollination, respectively) and high pollen fertility (average of 87.05 %). It was easy to develop new type B. napus by crossing the hexaploid with 142 lines of B. rapa from three ecotype groups, with the average crossability of 9.24 seeds per pod. The genetic variation of new type B. napus was diverse from that of current B. napus, especially in the A subgenome, revealed by genome-specific simple sequence repeat markers. Our data suggest that the strategy proposed here is a large-scale and highly efficient method to introgress genomic components of B. rapa into B. napus.