Reducing environmental impacts and improving N utilization are critical to ensuring food security in China.  Although root-zone fertilization has been considered an effective strategy to improve nitrogen use efficiency (NUE), the effect of controlled-release urea (CRU) applied in conjunction with normal urea in this mode is unclear.  Therefore, a 3-year field experiment was conducted using a no-N-added as a control and two fertilization modes (FF, furrow fertilization by manual trenching, i.e., farmer fertilizer practice; HF: root-zone hole fertilization by point broadcast manually) at 210 kg N ha^–1 (controlled-release:normal fertilizer=5:5), along with a 1-year in-situ microplot experiment.  Maize yield, NUE and N loss were investigated under different fertilization modes.  The results showed that compared with FF, HF improved the average yield and N recovery efficiency by 8.5 and 22.3% over three years, respectively.  HF had a greater potential for application than FF treatment, which led to increases in dry matter accumulation, total N uptake, SPAD value and LAI.  In addition, HF remarkably enhanced the accumulation of ¹⁵N derived from fertilizer by 17.2% compared with FF, which in turn reduced the potential loss of ¹⁵N by 43.8%.  HF increased the accumulation of N in the tillage layer of soils at harvest for potential use in the subsequent season relative to FF.  Hence, HF could match the N requirement of summer maize, sustain yield, improve NUE and reduce environmental N loss simultaneously.  Overall, root-zone hole fertilization with blended CRU and normal urea can represent an effective and promising practice to achieve environmental integrity and food security on the North China Plain, which deserves further application and investigation.

Photosynthesis occurs mainly in chloroplasts, whose development is regulated by proteins encoded by nuclear genes.  Among them, pentapeptide repeat (PPR) proteins participate in organelle RNA editing.  Although there are more than 450 members of the PPR protein family in rice, only a few affect RNA editing in rice chloroplasts.  Gene editing technology has created new rice germplasm and mutants, which could be used for rice breeding and gene function study.  This study evaluated the functions of OsPPR9 in chloroplast RNA editing in rice.  The osppr9 mutants were obtained by CRISPR/Cas9, which showed yellowing leaves and a lethal phenotype, with suppressed expression of genes associated with chloroplast development and accumulation of photosynthetic-related proteins.  In addition, loss of OsPPR9 protein function reduces the editing efficiency of rps8-C182, rpoC2-C4106, rps14-C80, and ndhB-C611 RNA editing sites, which affects chloroplast growth and development in rice.  Our data showed that OsPPR9 is highly expressed in rice leaves and encodes a DYW-PPR protein localized in chloroplasts.  Besides, the OsPPR9 protein was shown to interact with OsMORF2 and OsMORF9.  Together, our findings provide insights into the role of the PPR protein in regulating chloroplast development in rice. 

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation.  African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.  Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China.  These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry.  They are difficult to be distinguished only by clinical diagnosis.  Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.  In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5´ UTR), African swine fever virus (ASFV) (B646L), and APPV (5´ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.  The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV).  The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL^–1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%.  Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively.  The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the  compliance results were the same (100%).  A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively.  This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

Sex determination in plants gives rise to unisexual flowers.  A better understanding of the regulatory mechanism underlying the production of unisexual flowers will help to clarify the process of sex determination in plants and allow researchers and farmers to harness heterosis.  Androecious cucumber (Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus reducing the cost of seed production.  The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding.  Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate (EMS)-mutagenized library of the cucumber inbred line ‘406’.  Genetic analysis, whole-genome resequencing, and molecular marker-assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11 (ACS11) conferred androecy.  The mutation caused an amino acid change from serine (Ser) to phenylalanine (Phe) at position 301 (S301F).  In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzymatic activity.  This study provides a new germplasm for use in cucumber breeding as the androecious male parent, and it offers new insights into the catalytic mechanism of ACS enzymes.

Numbers of vertebrae is an important economic trait associated with body size and meat productivity in animals.  However, the genetic basis of vertebrae number in donkey remains to be well understood.  The aim of this study was to identify candidate genes affecting the number of thoracic (TVn) and the number of lumbar vertebrae (LVn) in Dezhou donkey.  A genome-wide association study was conducted using whole genome sequence data imputed from low-coverage genome sequencing.  For TVn, we identified 38 genome-wide significant and 64 suggestive SNPs, which relate to 7 genes (NLGN1, DCC, SLC26A7, TOX, WNT7A, LOC123286078, and LOC123280142).  For LVn, we identified 9 genome-wide significant and 38 suggestive SNPs, which relate to 8 genes (GABBR2, FBXO4, LOC123277146, LOC123277359, BMP7, B3GAT1, EML2, and LRP5).  The genes involve in the Wnt and TGF-β signaling pathways and may play an important role in embryonic development or bone formation and could be good candidate genes for TVn and LVn.

As important yield-related traits, thousand-grain weight (TGW), grain number per spike (GNS) and grain weight per spike (GWS) are crucial components of wheat production.  To dissect their underlying genetic basis, a double haploid (DH) population comprised of 198 lines derived from 8762/Keyi 5214 was constructed.  We then used genechip to genotype the DH population and integrated the yield-related traits TGW, GNS and GWS for QTL mapping.  Finally, we obtained a total of 18 942 polymorphic SNP markers and identified 41 crucial QTLs for these traits.  Three stable QTLs for TGW were identified on chromosomes 2D (QTgw-2D.3 and QTgw-2D.4) and 6A (QTgw-6A.1), with additive alleles all from the parent 8762, explaining 4.81–18.67% of the phenotypic variations.  Five stable QTLs for GNS on chromosomes 3D, 5B, 5D and 6A were identified.  QGns-5D.1 was from parent 8762, while the other four QTLs were from parent Keyi 5214, explaining 5.89–7.08% of the GNS phenotypic variations.  In addition, a stable GWS genetic locus QGws-4A.3 was detected from the parent 8762, which explained 6.08–6.14% of the phenotypic variations.  To utilize the identified QTLs, we developed STARP markers for four important QTLs, Tgw2D.3-2, Tgw2D.4-1, Tgw6A.1 and Gns3D.1.  Our results provide important basic resources and references for the identification and cloning of genes related to TGW, GNS and GWS in wheat.

Salmonella is one of the most common food-borne pathogens and its resistance in chicken can be improved through genetic selection.  The heterophils/lymphocytes (H/L) ratio in the blood reflects the immune system status of chicken.  We compared the genome data and spleen transcriptomes between the H/L ratio-selected and non-selected chickens, after Salmonella infection, aiming to identify the key genes participating in the antibacterial activity in the spleen.  The results revealed that, the selected population had stronger (P<0.05) liver resistance to Salmonella typhimurium (ST) than the non-selected population.  In the selected and non-selected lines, the identified differentiation genes encode proteins involved in biological processes or metabolic pathways that included the TGF-beta signaling pathway, FoxO signaling pathway, and Salmonella infection pathway.  The results of the analysis of all identified differentially expressed genes (DEGs) of spleen revealed that the G protein-coupled receptor (GPCR) and insulin-like growth factor (IGF-I) signaling pathways were involved in the Salmonella infection pathway.  Integrated analysis of DEGs and F_ST (fixation index), identified candidate genes involved in Salmonella infection pathway, such as GPR39, NTRK2, and ANXA1.  The extensive genomic changes highlight the polygenic genetic of the immune response in these chicken populations.  Numerous genes related to the immune performance are differentially expressed in the selected and non-selected lines and the selected lines has a higher resistance to Salmonella. 

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants.  In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall.  The function of another major laccase gene, StLAC6, was studied here.  The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal.  The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains.  In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes.  This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.   

Obesity presents a serious threat to human health and broiler performance.  The expansion of adipose tissue is mainly regulated by the differentiation of preadipocytes.  The differentiation of preadipocytes is a complex biological process regulated by a variety of transcription factors and signaling pathways.  Previous studies have shown that the transcription factor HMG-box protein 1 (HBP1) can regulate the differentiation of mouse 3T3-L1 preadipocytes by activating the Wnt/β-catenin signaling pathway.  However, it is unclear whether HBP1 involved in chicken preadipocyte differentiation and which signaling pathways it regulates.  The aim of the current study was to explore the biological function and molecular regulatory mechanism of HBP1 in the differentiation of chicken preadipocytes.  The expression patterns of chicken HBP1 in abdominal adipose tissue and during preadipocyte differentiation were analyzed by RT-qPCR and Western blot.  The preadipocyte stably overexpressing HBP1 or knockout HBP1 and their control cell line were used to analyze the effect of HBP1 on preadipocyte differentiation by oil red O staining, RT-qPCR and Western blot.  Cignal 45-Pathway Reporter Array was used to screen the signal pathways that HBP1 regulates in the differentiation of chicken preadipocytes.  Chemical inhibitor and siRNA for signal transducer and activator of transcription 3 (STAT3) were used to analyze the effect of STAT3 on preadipocyte differentiation.  The preadipocyte stably overexpressing HBP1 was transfected by the siRNA of STAT3 or treated with a chemical inhibitor of STAT3 for the rescue experiment.  The results of gene expression analysis showed that the expression of HBP1 was related to abdominal fat deposition and preadipocyte differentiation in chickens.  The results of function gain and loss experiments indicated that overexpression/knockout of HBP1 in chicken preadipocytes could inhibit/promote (P<0.05) lipid droplet deposition and the expression of adipogenesis-related genes.  Mechanismlly, HBP1 activates (P<0.05) the signal transducer and activator of transcription 3 (STAT3) signaling pathway by targeting janus kinase 2 (JAK2) transcription.  The results of functional rescue experiments indicated that STAT3 signaling mediated the regulation of HBP1 on chicken preadipocyte differentiation.  In conclusion, HBP1 inhibits chicken preadipocyte differentiation by activating the STAT3 signaling pathway via directly enhancing JAK2 expression.  Our findings provided new insights for further analysis of the molecular genetic basis of chicken adipose tissue growth and development.

Rose is one of the most important ornamental and economic plants in the world.  Modern rose cultivars are primarily tetraploid, and during meiosis, they may exhibit double reduction or preferential chromosome pairing.  Therefore, the construction of a high density genetic map of tetraploid rose is both challenging and instructive.  In this study, a tetraploid rose population was used to conduct a genetic analysis using genome sequencing.  A total of 17 382 single nucleotide polymorphism (SNP) markers were selected from 2 308 042 detected SNPs.  Combined with 440 previously developed simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) markers, a marker dosage of 6 885 high quality markers was successfully assigned by GATK software in the tetraploid model.  These markers were used in the construction of a high density genetic map, containing the expected seven linkage groups with 6 842 markers, a total map length of 1 158.9 cM, and an average inter-marker distance of 0.18 cM.  Quantitative trait locus (QTL) analysis was subsequently performed to characterize the genetic architecture of petal number and flower diameter.  One major QTL (qpnum-3-1) was detected for petal number in three consecutive years, which explained 20.18–22.11% of the variation in petal number.  Four QTLs were detected for flower diameter; the main locus, qfdia-2-2, was identified in two consecutive years.  Our results will benefit the molecular marker-assisted breeding of modern rose cultivars.  In addition, this study provides a guide for the genetic and QTL analysis of autotetraploid plants using sequencing-based genotyping methods.