Utilizing resequencing big data to facilitate Brassica vegetable breeding:tracing introgression pedigree and developing highly specific markers for clubroot resistance

Clubroot caused by Plasmodiophora brassicae is a devastating disease of Cruciferous crops.Developing cultivars with clubroot resistance(CR)is the most effective control measure.For the two major Brassica vegetable species B.rapa and B.oleracea,several commercial cultivars with unclear CR pedigrees have been intensively used as CR donors in breeding.However,the continuous occurrence of CR-breaking makes the CR pedigree underlying these cultivars one of the breeders'most urgent concerns.The complex intraspecific diversity of these two major Brassica vegetables has also limited the applicability of CR markers in different breeding programs.Here we first traced the pedigree underlying two kinds of CR that have been widely applied in breeding by linkage and introgression analyses based on public resequencing data.In B.rapa,a major locus CRzi8 underlying the CR of the commercial CR donor‘DegaoCR117’was identified.CRzi8 was further shown to have been introgressed from turnip(B.rapa ssp.rapifera)and that it carried a potential functional allele of Crr1a.The turnip introgression carried CRb^(c),sharing the same coding sequence with the CRb that was also identified from chromosome C07 of B.oleracea CR cultivars with different morphotypes.Within natural populations,variation analysis of linkage intervals of CRzi8,PbBa8.1,CRb,and CRb^(c)yielded easily resolved InDel markers(>20 bp)for these fundamental CR genes.The specificity of these markers was tested in diverse cultivars panels,and each exhibited high reliability in breeding.Our research demonstrates the value of the practice of applying resequencing big data to solve urgent concerns in breeding programs.

Restored intestinal integrity,nutrients transporters,energy metabolism,antioxidative capacity and decreased harmful microbiota were associated with IUGR piglet's catch-up growth before weanling

Background:Intrauterine growth restriction(IUGR)is a major inducer of higher morbidity and mortality in the pig industry and catch-up growth(CUG)before weanling could significantly restore this negative influence.But there was limited knowledge about the underlying mechanism of CUG occurrence.Methods:Eighty litters of newborn piglets were divided into normal birth weight(NBW)and IUGR groups according to birth weight.At 26 d,those piglets with IUGR but over average body weight of eighty litters of weaned piglets were considered as CUG,and the piglets with IUGR still below average body weight were considered as NCUG.This study was conducted to systemically compare the intestinal difference among NBW,CUG and NCUG weaned piglets considering the crucial role of the intestine for piglet growth.Results:The results indicated that the m RNA expression of nutrients(amino acids,glucose,and fatty acids)transporters,and mitochondrial electron transport chain(ETC)I were upregulated in CUG piglets'gut with improved morphology compared with those NCUG,as well as the ratio of P-AMPK/AMPK protein expression which is the indicator of energy metabolism.Meanwhile,CUG piglet's gut showed higher antioxidative capacity with increased SOD and GSHPx activity,decreased MDA levels,as well as higher m RNA expressions of Nrf2,Keap1,SOD,and GSH-Px.Furthermore,inflammatory parameters including TNF-α,IL-1β,IL-6,and IL-12 factors,and the activation of MAPK and NF-κB signaling pathways were significantly elevated in the NCUG intestine,while the protein expression of ZO-1,Occludin and Claudin-1 was reduced.The alpha diversity of fecal microbiota was higher in CUG piglets in contrast with NCUG piglets,and the increased beneficial bacteria and decreased pathogenic bacteria was also observed in CUG piglets.Conclusions:CUG piglet's intestine showed comprehensive restoration including higher nutrients transport,energy metabolism,antioxidant capacity,and intestinal physical barrier,while lower oxidative stress,inflammatory response,and pathogenic microbiota.

Altered metabolic state impedes limb regeneration in salamanders

Salamanders are unique among tetrapods in their ability to regenerate the limbs throughout life.Like other poikilothermic amphibians,salamanders also show a remarkable capacity to survive long periods of starvation.Whether the physiological reserves necessary for tissue regeneration are preserved or sacrificed in starved salamanders is unknown.In the current study,we maintained Iberian ribbed newts(Pleurodeles waltl)under extreme physiological stress to assess the extent of regeneration and identify the molecular and cellular changes that may occur under such conditions.After 19 months of complete food deprivation,the animals exhibited extensive morphological and physiological adaptations but remained behaviorally active and vigilant.Autophagy was elevated in different tissues and the transformed gut microbiota indicated remodeling of the intestinal tract related to autophagy.Upon limb amputation in animals starved for 21 months,regeneration proceeded with progenitor cell proliferation and migration,leading to limb blastema formation.However,limb outgrowth and patterning were substantially attenuated.Blockage of autophagy inhibited cell proliferation and blastema formation in starved animals,but not in fed animals.Hence,tissue autophagy and the regenerative response were tightly coupled only when animals were under stress.Our results demonstrate that under adverse conditions,salamanders can exploit alternative strategies to secure blastema formation for limb regeneration.

Recent advances in nano-enabled agriculture for improving plant performance

Nano-enabled agriculture is an emerging hot topic.To facilitate the development of nano-enabled agriculture,reviews addressing or discussing the applications,knowledge gap,future research needs,and possible new research field of plant nanobiotechnology in agricultural production are encouraged.Here we review the following topics in plant nanobiotechnology for agriculture:1)improving stress tolerance,2)stress sensing and early detection,3)targeted delivery and controlled release of agrochemicals,4)transgenic events in non-model crop species,and 5)seed nanopriming.We discuss the knowledge gaps in these topics.Besides the use of nanomaterials for harvesting more electrons to improve photosynthetic performance,they could be used to convert n IR and UV to visible light to expand the light spectrum for photosynthesis.We discuss this approach to maintaining plant photosynthesis under light-insufficient conditions.Our aim in this review is to aid researchers to learn quickly how to use plant nanobiotechnology for improving agricultural production.

A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish

Zebrafish(Danio rerio)has been used as a promising animal model to study gonadal development and gametogenesis.Although previous studies have identified critical molecules participating in zebrafish gonad differentiation,a landscape view of the biological processes involved in this process is still lacking.Here we isolated intact zebrafish differentiating gonads,at 25 days post-fertilization(dpf)and 30 dpf and conducted RNA-seq analyses on the juvenile gonads that tended to develop into ovaries or testes.Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the biological process level.During ovary differentiation,the biological processes related to metabolic activities in the production of energy and maternal substances,RNA degradation,and DNA repair were enriched.During testis differentiation,the biological processes related to cell proliferation,differentiation,and morphogenesis were enriched,with a total of 15 signaling pathways.Notably,we reveal that the immune-related processes are extensively involved in the regulation of testis development.Overall,this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.

A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases

Clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9(Cas9)-mediated gene editing is revolutionizing plant research and crop breeding.Here,we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small-to large-scale genome editing in plants.This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs(gRNAs)(FLASH),and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time.The identities of gRNAs in Agrobacterium mixtures and trans-genic plants can therefore be read out by detecting the FLASH tags,a process that requires only conven-tional PCR and gel electrophoresis rather than sequencing.We generated an arrayed CRISPR library target-ing all 1,072 members of the receptor-like kinase(RLK)family in rice.One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs,and 74.3%(710/955)of the target genes had three or more independent T0 lines.Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly(92.1%)associated with frameshift mutations in the target genes.In addition,the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes.Furthermore,we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes.This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.

High-efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR-Cas systems

Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products,abundant thermophilic enzymes,and promising fermentation capacities.However,efficient and versatile genome editing tools are not available for this species.In this study,we developed an efficient genome editing tool for T.thermophilus HB27 based on its endogenous type IB,I-C,and III-A/B CRISPR-Cas systems.First,we systematically characterized the DNA interference capabilities of the different types of the native CRISPR-Cas systems in T.thermophilus HB27.We found that genomic manipulations such as gene deletion,mutation,and in situ tagging could be easily implemented by a series of genome-editing plasmids carrying an artificial self-targeting mini-CRISPR and a donor DNA responsible for the recombinant recovery.We also compared the genome editing efficiency of different CRISPR-Cas systems and the editing plasmids with donor DNAs of different lengths.Additionally,we developed a reporter gene system for T.thermophilus based on a heat-stableβ-galactosidase gene TTP0042,and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.

The CropGPT project:Call for a global,coordinated effort in precision design breeding driven by Al using biological big data

Since the beginning of humanity,people have been seeking practical solutions to meet their most basic need:food.The advent of agriculture greatly increased the food supply and led to a rapid increase in the human population.Enhanced food production via agriculture can be attributed to both improved crop management practices and crop breeding through genetics.Plant(crop)breeding has been categorized into four major stages based on the techniques employed(Wallace et al.,2018).

Rapid detection of fluoroquinolone residues in aquatic products based on a gold-labeled microwell immunochromatographic assay

Objectives:Fluoroquinolones(FQs)are widely used in aquaculture,and their residues have caused many problems threatening human health.Here,this study aims to develop a colloidal gold immunochromatographic strip based on gold-labeled microwells to screen the residues of FQs on site.Materials and methods:The Protein A Magarose Beads affinity chromatography method was adopted to purify the ascites against FQs.By using a strategy of heterologous coating antigen,different coating antigens are applied to detect FQs.The gold-labeled microwell immunochromatographic assay was used to improve the sensitivity of the test strip by the advanced reaction of antigen and antibody.Results:The antibodies were verified to be of high purity up to 99%,and the titer reached 1:1024000.The combination(enoxacin-OVA and the antibody)detected the 4 banned FQs(pefloxacin,PEF;norfloxacin,NOR;lomefloxacin,LOM;ofloxacin,OFL)with the 50%inhibiting concentration(IC50)values ranging from 1.3 to 2.1 ng/mL and cross-reactions ranging from 67.3%to 106.1%.The analysis of spiked crucian carp,silver carp,grass carp,and shrimp samples showed that the limit of detection for PEF,NOR,LOM,and OFL was 4μg/kg.A comparative study with liquid chromatography tandem mass spectrometry(LC-MS/MS)demonstrated that the assay provided an effective screening tool for the rapid detection of FQs residues.Conclusions:The results indicated that the test strip can realize full coverage recognition of the 4 banned FQs and has good accuracy,specifi-city,reproducibility,and stability;therefore,they are more suitable for rapid detection of FQs in aquatic products.

Transcription factor CsESE3 positively modulates both jasmonic acid and wax biosynthesis in citrus

PLIP lipases can initiate jasmonic acid(JA)biosynthesis.However,little is known about the transcriptional regulation of this process.In this study,an ERF transcription factor(CsESE3)was found to be co-expressed with all necessary genes for JA biosynthesis and several key genes for wax biosynthesis in transcriptomes of‘Newhall’navel orange.CsESE3 shows partial sequence similarity to the well-known wax regulator SHINEs(SHNs),but lacks a complete MM protein domain.Ectopic overexpression of CsESE3 in tomato(OE)resulted in reduction of fruit surface brightness and dwarf phenotype compared to the wild type.The OE tomato lines also showed significant increases in the content of wax and JA and the expression of key genes related to their biosynthesis.Overexpression of CsESE3 in citrus callus and fruit enhanced the JA content and the expression of JA biosynthetic genes.Furthermore,CsESE3 could bind to and activate the promoters of two phospholipases from the PLIP gene family to initiate JA biosynthesis.Overall,this study indicated that CsESE3 could mediate JA biosynthesis by activating PLIP genes and positively modulate wax biosynthesis.The findings provide important insights into the coordinated control of two defense strategies of plants represented by wax and JA biosynthesis.

GenOrigin: A comprehensive protein-coding gene origination database on the evolutionary timescale of life

The origination of new genes contributes to the biological diversity of life. New genes may quickly build their network, exert important functions, and generate novel phenotypes. Dating gene age and inferring the origination mechanisms of new genes, like primate-specific genes, is the basis for the functional study of the genes. However, no comprehensive resource of gene age estimates across species is available. Here,we systematically date the age of 9,102,113 protein-coding genes from 565 species in the Ensembl and Ensembl Genomes databases, including 82 bacteria, 57 protists, 134 fungi, 58 plants, 56 metazoa, and 178 vertebrates, using a protein-family-based pipeline with Wagner parsimony algorithm. We also collect gene age estimate data from other studies and uniformly distribute the gene age estimates to time ranges in a million years for comparison across studies. All the data are cataloged into Gen Origin(http://genorigin.chenzxlab.cn/), a user-friendly new database of gene age estimates, where users can browse gene age estimates by species, age, and gene ontology. In Gen Origin, the information such as gene age estimates,annotation, gene ontology, ortholog, and paralog, as well as detailed gene presence/absence views for gene age inference based on the species tree with evolutionary timescale, is provided to researchers for exploring gene functions.

Recent strategies of collagen-based biomaterials for cartilage repair: from structure cognition to function endowment

Collagen,characteristic in biomimetic composition and hierarchical structure,boasts a huge potential in repairing cartilage defect due to its extraordinary bioactivities and regulated physicochemical properties,such as low immunogenicity,biocompatibility and controllable degradation,which promotes the cell adhesion,migration and proliferation.Therefore,collagen-based biomaterial has been explored as porous scaffolds or functional coatings in cell-free scaffold and tissue engineering strategy for cartilage repairing.Among those forming technologies,freeze-dry is frequently used with special modifications while 3D-printing and electrospinning serve as the structure-controller in a more precise way.Besides,appropriate cross-linking treatment and incorporation with bioactive substance generally help the collagen-based biomaterials to meet the physicochemical requirement in the defect site and strengthen the repairing performance.Furthermore,comprehensive evaluations on the repair effects of biomaterials are sorted out in terms of in vitro,in vivo and clinical assessments,focusing on the morphology observation,characteristic production and critical gene expression.Finally,the challenge of biomaterial-based therapy for cartilage defect repairing was summarized,which is,the adaption to the highly complex structure and functional difference of cartilage.

Salicylic acid inhibits gibberellin signaling through receptor interactions

It is well known that plants activate defense responses at the cost of growth.However,the underlying molecular mechanisms are not well understood.The phytohormones salicylic acid(SA)and gibberellin(GA)promote defense response and growth,respectively.Here we show that SA inhibits GA signaling to repress plant growth.We found that the SA receptor NPR1 interacts with the GA receptor GID1.Further biochemical studies revealed that NPR1 functions as an adaptor of ubiquitin E3 ligase to promote the polyubiquitination and degradation of GID1,which enhances the stability of DELLA proteins,the negative regulators of GA signaling.Genetic analysis suggested that NPR1,GID1,and DELLA proteins are all required for the SA-mediated growth inhibition.Collectively,our study not only uncovers a novel regulatory mechanism of growth-defense trade-off but also reveals the interaction of hormone receptors as a new mode of hormonal crosstalk.