3D genome organization and its study in livestock breeding

Eukaryotic genomes are hierarchically packaged into cell nucleus,affecting gene regulation.The genome is organized into multiscale structural units,including chromosome territories,compartments,topologically associating domains(TADs),and DNA loops.The identification of these hierarchical structures has benefited from the development of experimental approaches,such as 3C-based methods(Hi-C,ChIA-PET,etc.),imaging tools(2D-FISH,3D-FISH,Cryo-FISH,etc.)and ligation-free methods(GAM,SPRITE,etc.).In recent two decades,numerous studies have shown that the 3D organization of genome plays essential roles in multiple cellular processes via various mechanisms,such as regulating enhancer activity and promoter-enhancer interactions.However,there are relatively few studies about the 3D genome in livestock species.Therefore,studies for exploring the function of 3D genomes in livestock are urgently needed to provide a more comprehensive understanding of potential relationships between the genome and production traits.In this review,we summarize the recent advances of 3D genomics and its biological functions in human and mouse studies,drawing inspiration to explore the 3D genomics of livestock species.We then mainly focus on the biological functions of 3D genome organization in muscle development and its implications in animal breeding.

Use of transcriptome sequencing to explore the effect of CSRP3 on chicken myoblasts

The mechanisms that regulate the specificity and maintenance of chicken muscle fiber types remain largely unknown. In mammals, CSRP3 has been shown to play a vital role in the maintenance of typical muscle structure and function. This study investigated the role that CSRP3 plays in chicken skeletal muscle. First, the antibody against chicken CSRP3 protein was prepared, and the expression levels of the mRNA and protein of the CSRP3 gene in four chicken skeletal muscles with different myofiber compositions were compared. Then the effects of CSRP3 silencing on the expression profile of chicken myoblast transcriptomes were analyzed. The results showed that the expression levels of the mRNA and protein of the CSRP3 gene were both associated with the composition of fiber types in chicken skeletal muscles. A total of 650 genes with at least 1.5-fold differences(Q<0.05) were identified, of which 255 genes were upregulated and 395 genes were downregulated by CSRP3 silencing. Functional enrichment showed that several pathways, including adrenergic signaling in cardiomyocytes, adipocytokine signaling pathway and apelin signaling pathway, were significantly(P<0.05) enriched both in differentially expressed genes and all expressed genes. The co-expressed gene network suggested that CSRP3 silencing caused a compensatory upregulation(Q<0.05) of genes related to the assembly of myofibrils, muscle differentiation, and contraction. Meanwhile, two fast myosin heavy chain genes(MyH1B and MyH1E)were upregulated(Q<0.05) upon CSRP3 silencing. These results suggested that CSRP3 plays a crucial role in chicken myofiber composition, and affects the distribution of chicken myofiber types, probably by regulating the expression of MyH1B and MyH1E.

Optimization of slow-release fertilizer application improves lotus rhizome quality by affecting the physicochemical properties of starch

To achieve the dual goals of high yield and good quality with low environmental costs,slow-release fertilizer(SRF)has been widely used in lotus cultivation as new type of fertilizer instead of traditional nitrogen fertilizer.However,the optimal amount of SRF and how it would promote lotus rhizome quality remain unclear.This study was designed to investigate the photosynthetic characteristics and the synthesis,accumulation,and physicochemical properties of lotus rhizome starches under six SRF levels(CK,S1,S2,S3,S4,and S5).Compared with CK(0 kg ha^(–1)),the net photosynthetic rate(P_(n))and SPAD values of leaves remained at higher levels under SRF treatment.Further research showed that SRF increased the lotus rhizome yield,the contents of amylose,amylopectin,and total starch,and the number of starch granules.Among the six SRF levels,S3(1035 kg ha^(–1))showed the greatest difference from CK and produced the highest levels.With the increasing SRF levels,the peak,hot and final viscosities decreased at first and then increased,but the setback viscosity and pasting temperature increased.In order to interpret these changes at the molecular level,the activities of key enzymes and relative expression levels of starch accumulation related genes were analyzed.Each of these parameters also increased under SRF treatment,especially under the S3 treatment.The results of this study show that SRF,especially S3(1035 kg ha^(–1)),is a suitable fertilizer option for lotus planting which can improve lotus rhizome quality by affecting starch accumulations related enzymes and genes.These results will be useful for SRF application to high-quality lotus rhizome production with low environmental costs.

CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells

The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide(MDP)-mediated inflammatory response of bovine rumen epithelial cells(BRECs).First,changes in the m RNA expression of proinflammatory factor genes in BRECs following 10μg m L^(–1)MDP treatments were examined.RT-q PCR results showed that the expression of pro-inflammatory factor(IL-1β,IL-6,and TNF-α)m RNAs were significantly increased under MDP stimulation(P<0.001).Moreover,SLC15A4-Knockout(SLC15A4-KO)cells were obtained through lentivirus packaging,transfection,screening,and cell monoclonal culture.In order to gain further insight into the potential function of SLC15A4,we utilized transcriptome data,which revealed a change in the genes between WT-BRECs and SLC15A4-KO.Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.Meanwhile,the down-regulated genes were mostly enriched in the nuclear factorκB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways.The results of RT-q PCR also verified these detected changes.To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses,we investigated the inflammatory responses of cells exposed to MDP.WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10μg m L^(–1)MDP,in comparison to a control without MDP.Our results show that SLC15A4-KO BRECs had reduced the expression of genes(IL-6,TNF-α,CXCL2,CXCL3,CXCL9,and CCL2)and proteins(p-p65 and p-p44/42)from the MDP-mediated inflammatory response compared to WT-BRECs(P<0.05).In this experiment,CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4,and its role was confirmed via the MDP-induced inflammatory response in BRECs.This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.

Dynamic chromatin architectures provide insights into the genetics of cattle myogenesis

Background Sharply increased beef consumption is propelling the genetic improvement projects of beef cattle in China.Three-dimensional genome structure is confirmed to be an important layer of transcription regulation.Although genome-wide interaction data of several livestock species have already been produced,the genome structure states and its regulatory rules in cattle muscle are still limited.Results Here we present the first 3D genome data in Longissimus dorsi muscle of fetal and adult cattle(Bos taurus).We showed that compartments,topologically associating domains(TADs),and loop undergo re-organization and the structure dynamics were consistent with transcriptomic divergence during muscle development.Furthermore,we annotated cis-regulatory elements in cattle genome during myogenesis and demonstrated the enrichments of promoter and enhancer in selection sweeps.We further validated the regulatory function of one HMGA2 intronic enhancer near a strong sweep region on primary bovine myoblast proliferation.Conclusions Our data provide key insights of the regulatory function of high order chromatin structure and cattle myogenic biology,which will benefit the progress of genetic improvement of beef cattle.

Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation

Follicle-stimulating hormone(FSH),an important hypothalamic-pituitary-gonadal axis(HPG)hormone,is secreted by the pituitary gland.This study confirms that FSH is expressed in chicken follicles at different stages,and positive FSHβ mRNA signals were stronger(P<0.05)in granulosa cells than in oocytes.The 369 bp coding sequence of FSHβ in ovaries is 100%identical to that in the pituitary gland.The experiment in vitro revealed that the ovary possessed FSH secretory capacity.Further,FSHβ mRNA was significantly upregulated(P<0.05)in follicles and significantly higher(P<0.05)than that in the pituitary gland by approximately 2–23 times with the development.The number of granulosa cells decreased significantly(P<0.05)in the cells with siRNA treatment,confirming that the ovarian FSH could promote granulosa cell proliferation.This view was supported by cell cycle analysis and CCND2 and CCNE2 expression.Further research indicated that no difference(P>0.05)was observed between the number of granulosa cells treated with FSHβ siRNA and in exogenous FSH.However,the number of granulosa cells without FSHβ siRNA transfection was significantly higher(P<0.05)for exogenous FSH.This finding suggests that the proliferative effect of exogenous FSH on ovarian granulosa cells depend on endogenous FSH.This study demonstrated that the FSH gene was expressed in chicken follicles and promoted ovarian granulosa cell proliferation,which enriched the theory on HPG axis.

Multi-environment QTL mapping of crown root traits in a maize RIL population

Crown root traits,including crown root angle(CRA),diameter(CRD),and number(CRN),are major determining factors of root system architecture,which influences crop production.In maize,the genetic mechanisms determining crown root traits in the field are largely unknown.CRA,CRD,and CRN were evaluated in a recombinant inbred line population in three field trials.High phenotypic variation was observed for crown root traits,and all measured traits showed significant genotype–environment interactions.Singleenvironment(SEA)and multi-environment(MEA)quantitative trait locus(QTL)analyses were conducted for CRA,CRD,and CRN.Of 46 QTL detected by SEA,most explained less than 10%of the phenotypic variation,indicating that a large number of minor-effect QTL contributed to the genetic component of these traits.MEA detected 25 QTL associated with CRA,CRD,and CRN,and 2 and 1 QTL were identified with significant QTL-by-environment interaction effects for CRA and CRD,respectively.A total of 26.1%(12/46)of the QTL identified by SEA were also detected by MEA,with many being detected in more than one environment.These findings contribute to our understanding of the phenotypic and genotypic patterns of crown root traits in different environments.The identified environment-specific QTL and stable QTL may be used to improve root traits in maize breeding.

Weighted gene co-expression network analysis identiffes potential regulators in response to Salmonella Enteritidis challenge in the reproductive tract of laying ducks

Salmonella Enteritidis(SE)is a zoonotic and vertically transmitted pathogen,often colonized in the reproductive tract of adult poultry,which can result in direct contamination of eggs and threaten human health.Previous studies have revealed that some pattern recognition receptors and resistance genes were involved in regulating immune responses to SE invasion in birds.However,the role of these immune response genes was not independent,and the interactions among the genes remained to be further investigated.In this study,SE burden and colonization were determined in reproductive tissue after the ducks were SE-infected,and RNA-sequencing was performed to construct co-expression networks by weighted gene co-expression network analysis(WGCNA).The result showed that SE could be isolated from 22% of infected-birds in any segment of the reproductive tract and the SE was readily colonized in the stroma,small follicle,isthmus,and vagina of the reproductive tracts in morbid ducks.The top central,highly connected genes were subsequently identified three specific modules in the above four tissues at the defined cut-offs(P<0.01),including 60 new candidate regulators and 125 transcription factors.Moreover,those 185 differentially expressed genes(DEGs)in these modules were co-expressed.Moreover,the hub genes(TRAF3,CXCR4 and IL13RA1)were identified to act with many other genes through immune response pathways including NF-kappaB,Toll-like receptor,steroid biosynthesis,and p53signaling pathways.These data provide references that will understand the immune regulatory relationships during SE infection,but also assist in the breeding of SE-resistant lines through potential biomarkers.

The expression of Lin28B was co-regulated by H3K4me2 and Wnt5a/β-catenin/TCF7L2

Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such information for Lin28B is limited. In this study, a fragment of the Lin28B promoter was cloned, the pEGFP-pLin28B vector was constructed. DF-1 chicken fibroblasts were transfected and the expression of green fluorescent protein (GFP) was measured. Furtherly, Lin28B promoter of different lengths fragments was cloned using the chromosome-walking method and the fragments were ligated into the PGL3-Basic vector, and transfected into DF-1 cells. Results of dual-luciferase reporter assay showed that the core of the Lin28B promoter was included in the sequence from –1 431 to –1 034 bp. The binding sites of the transcription factor TCF7L2 was showed within this sequence by bioinformatics analysis. The promoter activity of Lin28B was downregulated (P<0.05) when the TCF7L2 binding site was mutated. Further experiments suggested that Lin28B promoter activity responded to the activation or inhibition of Wnt signaling. Results of chromatin immunoprecipitation and quantitative PCR showed that β-catenin-TCF7L2 may be enriched in the Lin28B promoter core area. In vivo and in vitro activation or inhibition of Wnt signaling significantly up- or down-regulated (P<0.05) Lin28B expression. H3K4me2 enriched in the promoter of Lin28B, which affected the regulation of Wnt signaling to Lin28B. In conclusion, our results showed that H3K4me2 and Wnt5a/β-catenin/TCF7L2 were the positive regulators of Lin28B expression. Findings of this study may lay a theoretical foundation for illuminating the mechanism underlying Lin28B expression.

NH4Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis

Excess ammonia(NH_(3))in the circulation of dairy animals can reduce animal health and the quality of products for human consumption.To develop effective prevention and treatment methods,it is essential to examine the molecular mechanisms through which excess NH_(3) may affect the mammary gland.The present study used bovine mammary epithelial cells(BMECs)to evaluate the effects of exogenous NH_(4)Cl on the abundance of circular RNAs(circRNAs)using high-throughput sequencing.Among the identified circRNAs,circ02771 was the most significantly upregulated by exogenous NH_(4)Cl(P<0.05),with a fold change of 4.12.The results of the apoptosis and proliferation assays,transmission electron microscopy,H&E staining,and immunohistochemistry revealed that circ02771 increased apoptosis and inflammation.A double luciferase reporter assay revealed that circ02771 targeted miR-194b,and the overexpression of circ02771(pcDNA-circ02771)reduced(P<0.05)the expression of miR-194b and led to apoptosis and inflammation.Circ02771 also enhanced the expression of transforming growth factor beta-induced factor homeobox 1(TGIF1),which is a target gene of miR-194b.Overall,this study suggests that the circ02771/miR-194b/TGIF1 axis plays a role in mediating the effects of NH_(4)Cl on BMECs.Therefore,this axis provides a novel target to help control hazards within the mammary gland from high circulating NH_(4)Cl levels.

UBE2I stimulates female gonadal differentiation in chicken(Gallus gallus)embryos

Without known analogous sex-determining factors like SRY(sex determining region Y)in mammals,the chicken(Gallus gallus)sex determination mechanism still remains unclear,which highly restricts the biological research on chicken development and poultry single-sex reproduction.Here we not only characterized a new female-biased gene UBE2I and identified the expression pattern by qRT-PCR,but also described the functional role of UBE2I in the gonadal development of chicken embryos.Results showed that UBE2I exhibited a female-biased expression pattern in the early stage of PGCs(primordial germ cells)in embryonic gonads and robust expression in ovaries of newborn chickens.Most importantly,we successfully developed an effective method to interfere or overexpress UBE2I in chicken embryos through the intravascular injection.The qRT-PCR analysis showed that the sex-related genes(FOXL2,CYP19A1 and HINTW)in females were upregulated(P<0.05)under the overexpression of UBE2I and the sex-related genes(SOX9,DMRT1 and WT1)in females were downregulated(P<0.05)after interfering UBE2I.Furthermore,the change of UBE2I expression was associated with the level of estradiol and its receptors(AR and ESR),which suggests that UBE2I is necessary to initiate the female-specific development in chickens.In conclusion,this work demonstrates that UBE2I is a crucial sex differentiation-related gene in the embryonic development of chickens,which provides insights for further understanding the mechanism of sex determination in chickens.

Simulation of greenhouse cucumber growth,water and nitrogen dynamics in areas with high groundwater(HG)levels using the HG EU-Rotate_N model

The fruit yield of cucumber are associated with high input of nitrogen,which poses a risk of pollution to the environment.The EU-Rotate_N model has been used widely for its high performance in the simulation of vegetable growth,water and nitrogen dynamics.However,whether the underground water level affects the performance of the EU-Rotate_N model is unclear.In this study,we modified the groundwater level algorithms to the original model and named the modified model'the HG EU-Rotate_N model'.Experiments over two years on greenhouse cucumber with four different nitrogen(N)treatments(N1-N4)were conducted in Jiangsu Province,China,which has a high groundwater level(area_(HG)).Both original and modified models were used to simulate cucumber growth,water movement and N fate.For the soil water content,the measured values were significantly larger than the simulated values of the original model(value_(O))and closer to those of the HG model(value_(HG));for the soil available nitrogen concentration(SNC),the measured values were significantly higher and lower than value_(O) in 0−10 cm and 10−30 cm soil layers,respectively,and were also closer to those of the values_(HG).The higher SNC in the 0-10 cm soil layer indicated that a high groundwater level might increase the upwards movement of water and nitrogen in the 0-30 cm soil layer.The root mean square error,Nash Sutcliffe Efficiency and difference values show that the HG model was more applicable for areaHG than the original model.In this study,the nitrogen dosage of the N3 treatment was sufficient to meet the requirements of cucumber growth,indicating that the fertilization recommendation according to nitrogen nutrient balance was applicable in this area.

Unraveling the role of phase engineering in tuning photocatalytic hydrogen evolution activity and stability

In this work,taking NiSe_(2)as a prototype to be used as cocatalyst in photocatalytic hydrogen evolution,we demonstrate that the crystal phase of NiSe_(2)plays a vital role in determining the catalytic stability,rather than activity.Theoretical and experimental results indicate that the phase structure shows negligible influence to the charge transport and hydrogen adsorption capacity.When integrating with carbon nitride(CN)photocatalyst forming hybrids(m-NiSe_(2)/CN and p-NiSe_(2)/CN),the hybrids show comparable photocatalytic hydrogen evolution rates(3.26μmol/h and 3.75μmol/h).Unlike the comparable catalytic activity,we found that phase-engineered NiSe_(2)exhibits distinct stability,i.e.,m-NiSe_(2)can evolve H_(2) steadily,but p-NiSe_(2)shows a significant decrease in catalytic process(∼57.1%decrease in 25 h).The factor leading to different catalytic stability can be ascribed to the different surface conversion behavior during photocatalytic process,i.e.,chemical structure of m-NiSe_(2)can be well preserved in catalytic process,but partial p-NiSe_(2)tends to be converted to NiOOH.

One-step preparation of a novel graphitic biochar/Cu^(0)/Fe_(3)O_(4) composite using CO_(2)-ambiance pyrolysis to activate peroxydisulfate for dye degradation

Herein,a one-step co-pyrolysis protocol was adopted for the first time to prepare a novel pyrogenic carbon-Cu^(0)/Fe_(3)O_(4)heteroatoms (FCBC) in CO_(2)ambiance to discern the roles of each component in PDS activation.During co-pyrolysis,CO_(2)catalyzed formation of reducing gases by biomass which facilitated reductive transformation of Fe^(3+)and Cu^(2+)to Cu^(0)and Fe_(3)O_(4),respectively.According to the analysis,the resulting metal (oxide) catalyzed graphitization of biocharand decomposition of volatile substances resulting in an unprecedented surface area (1240 m^(2)/g).The resulting FCBC showed greater structural defects and less electrical impedance.Batch experiments indicated that Rhodamine B (RhB) degradation by FCBC (100%) was superior to Fe_(3)O_(4)(50%) and Cu^(0)/Fe_(3)O_(4)(76.4%) in persulfate (PDS) system,which maintained reasonable efficiency (75.6%-63.6%) within three cycles.The reactive oxygen species (ROS) associated with RhB degradation was identified by an electron paramagnetic resonance and confirmed by scavenging experiments.RhB degradation invoked both(sulfate and dominantly hydroxyl) radical and non-radical (singlet oxygen,^(1)O_(2)) pathways.Regarding FCBC,Cu^(0)can continuously react with Fe^(3+)in Fe_(3)O_(4)to generate larger quantities of Fe^(2+),and both Cu^(0)and Fe^(2+)activated PDS to yield sulfate radicals which was quickly converted to hydroxyl radical.Besides,Cu^(0)/Cu^(2+)could complex with PDS to form a metastable complex,which particularly contributed to1O_(2)generation.These cascade reactions by FCBC were reinforced by carbonyl group of biochar and favorable electron transfer ability.This work highlighted a new approach to prepare a magnetic and environment-benign heterogonous catalyst to remove organic pollutants in water.

Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

Modes of genetic adaptations underlying functional innovations in the rumen

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers.We analyzed 897 transcriptomes from three Cetartiodactyla lineages:ruminants,camels and cetaceans,as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen functional innovations.We identified genes with relatively high expression in the rumen,of which many appeared to be recruited from other tissues.These genes show functional enrichment in ketone body metabolism,regulation of microbial community,and epithelium absorption,which are the most prominent biological processes involved in rumen innovations.Several modes of genetic change underlying rumen functional innovations were uncovered,including coding mutations,genes newly evolved,and changes of regulatory elements.We validated that the key ketogenesis rate-limiting gene(HMGCS2)with five ruminant-specific mutations was under positive selection and exhibits higher synthesis activity than those of other mammals.Two newly evolved genes(LYZ1 and DEFB1)are resistant to Gram-positive bacteria and thereby may regulate microbial community equilibrium.Furthermore,we confirmed that the changes of regulatory elements accounted for the majority of rumen gene recruitment.These results greatly improve our understanding of rumen evolution and organ evo-devo in general.

Plant Genome Editing Using FnCpfl and LbCpf Nucleases at Redefined and Altered PAM-Sites

Dear Editor CRISPR from Prevotella and Francisella 1 （Cpfl） is an emerging RNA-guided endonuclease system that relies on thymidine-rich protospacer adjacent motif （PAM） for DNA targeting （Zetsche et al., 2015）. CRISPR-Cpfl has unique features that could be advantageous over the CRISPR-Cas9 system. For example, Cpfl requires only a 42 nt crRNA, while Cas9 uses 100 nt gRNA. While Cas9 generates blunt ends of DNA breaks, the Cpfl cleavage results in 5＇ overhangs distal from the protospacer, which may improve efficiency for NHEJ-based gene insertion. Interestingly, Cpfl proteins also have RNase activity （Fonfara et al., 2016）, which was utilized to process crRNA arrays for multiplexed genome editing in both mammalian systems and plants （Wang et al., 2017; Zetsche et al., 2017）.