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    Crop Genetics & Breeding·Germplasm Resources·Biotechnology

  • YANG Wenlong, LI Xixiang, ZHANG Xiaohui, SONG Jiangping, JIA Huixia, WANG Haiping
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    Gibberellin pathway is an important pathway in plant flowering regulation.In order to understand the role of gibberellin pathway related genes in the regulation of radish flowering. The structure,physicochemical properties,chromosome distribution,promoter cis-elements and tissue-specific expression of gibberellin biosynthesis and signal transduction related genes in radish were analyzed by bioinformatics.The expression levels of these genes in radish varieties with different florescence were detected by Real-time fluorescence quantitative PCR(qPCR).The results showed there were 46 genes related to gibberellin biosynthesis and signal transduction in radish genome,among them,the gene numbers of CPS,KS,KO,KAO,GA20OX,GA3OX,GA2OX,GAI,RGA,RGL,GID1 and SKP2 were 2,1,2,2,9,5,12,1,1,4,3 and 4,respectively.They were unevenly distributed on 9 chromosomes,molecular weight of their coding proteins were 21.32—127.80 ku,and the isoelectric points of the proteins were from 4.72 to 9.04.The analysis of gene structure and conserved domain showed that the number of exons of these 46 genes ranged from 1 to 21,and some conserved motifs were shared by most genes.Promoter cis-elements analysis showed that the promoters of these 46 genes contained cis-elements related to light,gibberellin,auxin,ABA,SA,low temperature,drought,etc.Using radish gene expression database analysis,it was found that the expression levels of these 46 genes were different in different tissues and at different developmental stages;qPCR detection showed that there were significant differences in the expression of these genes between early flowering material Xinlimei and late flowering material wild radish,suggesting that they may be closely related to the reproductive growth of radish.

  • SONG Pingli, LI Gang, XU Jianfeng, MA Qingcui, QI Baoxiu, ZHANG Yuxing
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    In order to clarify the biological function of gibberellin receptor GID1 in Pyrus betulifolia,and provide a good foundation for future development of P.betulifolia dwarf rootstocks using CRISPR/Cas9 gene editing technology.Pyrus betulifolia was used as the test material,and the PbGID1s genes were obtained by homologous cloning method.Bioinformatics analysis software was used to construct the gene structure and design the target sites;construction of sgRNA expression cassettes with targets into CRISPR/Cas9 expression vectors,through the mediation of Agrobacterium,the CRISPR/Cas9 expression vector was transferred into the cotyledons of P.betulifolia.Results showed that four PbGID1s were successfully cloned from P.betulifolia plants and named as PbGID1b-1,PbGID1b-2,PbGID1c-1 and PbGID1c-2. They all consisted of two exons and one intron found by gene structure analysis.Amino acid sequence comparison showed that all PbGID1s had the HGG and GXSXG conserved domains.Five gRNAs that could potentially edit all 4 PbGID1s simultaneously were successfully constructed into a single CRISPR/Cas9 vector,pYLCRISPR/Cas9P35S-N.The results of the genetic transformation test of P.betulifolia showed that a total of 595 cotyledons of P.betulifolia were infiltrated,176 resistant buds and 33 positive plantlets were obtained,and the transformation efficiency reached 5.55%.A CRISPR/Cas9 vector was successfully constructed that could simultaneously target the PbGID1s family genes of P.betulifolia.Through the mediation of Agrobacterium,the vector was successfully transformed into P.betulifolia cotyledons,and positive plants were obtained.

  • CHEN Na, SHAO Qin, LI Xiaopeng, GAO Yang, LU Qineng
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    In order to explore the important role of SBP1(S-RNase-binding protein 1)gene in plant self-incompatibility,a diploid wild potato was used as the material to obtain the full-length cDNA sequence of potato SpSBP1 (GenBank: MZ803088)by using RT-PCR cloning technique,and performed bioinformatics analysis and CRISPR/Cas9 vector construction for SpSBP1 gene.The results showed that the full length cDNA of SpSBP1 gene was 1 176 bp,containing 92 bp 5' non-coding region and 163 bp 3' non-coding region.The maximum open reading frame(ORF)of SpSBP1 gene was 921 bp,which encoding 306 amino acids.Protein domain analysis showed that SpSPB1 protein included Smc superfamily domain,RING finger domain and Zinc finger domain.The homologous sequence alignment and phylogenetic tree analysis showed that SpSBP1 had the highest homology with S.chacoense,followed by Nicotiana alata.Bioinformatics analysis showed that the molecular weight of SpSBP1 was 34.731 44 ku,with a theoretical isoelectric point of 5.10,and it was speculated that SpSBP1 was an acid unstable hydrophilic protein. The secondary structure predicted that the protein was mainly composed of α helix and random curling. And the protein was a non-secretory protein and had no transmembrane structure. SpSBP1 gene was cloned from wild diploid potato,and CRISPR/Cas9 vector was successfully constructed,and also the genetic transformation was carried out.

  • ZHANG Xi, QIAN Yuyuan, WANG Guang'en, LIU Yi, QUAN Yuewei, CUI Shufang, MI Huanfang, LI Junlan
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    In order to explore the function of GhERF105-like in the development of pigment glands or gossyol metabolism of cotton,the GhERF105-like gene of Zhongmiansuo 12 was cloned by RT-PCR,and the bioinformatics analysis and expression patterns were analyzed.The results showed that the coding region of GhERF105-like was 711 bp,encoding 236 amino acids.The molecular weight of GhERF105-like was 26.3 ku,and the theoretical pI was 7.72.The predicted secondary structure of GhERF105-like was 22.88% α-helix,13.14% extended strand,3.81% β-turn and 60.17% random coil.GhERF105-like had an AP2 domain at 91—155 aa.Two threonine,one tyrosine and one serine phosphorylation sites were predicted to be located in the AP2 domain.Phylogenetic analysis showed that motifs of GhERF105-like and its homologous proteins were highly conserved in different species.GhERF105-like motifs were consistent with homologous proteins in Gossypium hirsutum,G.mustelinum,G.darwinii,G.raimondii and G.barbadense.The expression of GhERF105-like in each tissue of glanded cotton Zhongmiansuo 12 was significantly or extremely significantly higher than that of dominant glandless Zhongmiansuo 12.The expression levels of GhERF105-like in leaves of different glanded cotton varieties/lines were different,and were all significantly higher than the expressions of dominant or recessive glandless cotton varieties/lines.GhERF105-like was significantly up-regulated by ABA and BR,but significantly or extremely significantly down-regulated by MeJA,Eth,NaCl and PEG treatment.In conclusion,GhERF105-like is closely related to the development of pigment glands in cotton,and may regulate gland development or gossypol metabolism through ABA,BR,Eth and JA pathways.

  • GU Yahui, ZHANG Changqing, ZHANG Xuebing, ZHANG Xinyue, ZHANG Xinjie, GE Rongchao
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    Abiotic stresses such as drought and soil salinization seriously affect the growth,development and yield of crops.GmRPK2 gene was cloned and constructed into the overexpression vector,then the vector was transformed into Arabidopsis thaliana and the homozygotes was screened.Under PEG,NaCl and ABA stress,the resistance of GmRPK2 overexpression Arabidopsis was tested.The results showed that the germination rate of the overexpressed Arabidopsis seeds was significantly lower than wild type.The elongation of the overexpressed Arabidopsis seedlings roots was also shorter than that of wild-type.In the stress resistance test of Arabidopsis plants,it was found that after salt stress and drought stress treatment,the inhibition of transgenic Arabidopsis plants was more obvious than that of wild-type Arabidopsis plants.The stress resistance test at adult stage showed that the growth inhibition of overexpressed Arabidopsis was more obvious under salt and drought stress.In order to explore the internal mechanism of the reduction of its stress resistance,the physiological indexes of GmRPK2 overexpressed Arabidopsis were detected.The results showed that,the chlorophyll content of overexpressed Arabidopsis was significantly lower,the malondialdehyde and conductivity were obviously higher,and the DAB staining was deeper than that of the wild type.After drought stress treatment,the contents of proline and soluble sugar of overexpressed Arabidopsis were obviously lower than those of wild type,and the water loss rate was obviously higher.The results of RT-PCR showed that GmRPK2 overexpressed in Arabidopsis obviously inhibited SAD1,P5CS1 and ADH1 expression.It was preliminarily speculated that it might inhibit the transmission of stress signals by inhibiting the expression of these genes.Therefore,GmRPK2 gene may affect the stress resistance of Arabidopsis by inhibiting CDPK stress resistance signal transduction pathway,reducing proline accumulation and weakening ABA signal transduction pathway.

  • ZHOU Zhenzhong, LUO Biao, LUO Ying, XIA Min, WANG Qiming, RAO Liqun
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    GlycosyltransferaseⅠ(GT Ⅰ)family is one of the glycosyltransferases,whose main functions involve the glycosyltransferase transfer of flavonoids and the synthesis of starch and sucrose.In order to reveal the role of GTⅠ gene family in the growth and development and high temperature tolerance of japonica rice, and to provide a basis for the functional study of GT Ⅰ gene family members in japonica rice,and provide ideas for rice variety improvement and high temperature tolerance. 30 members of GT Ⅰ gene family were identified by bioinformatics methods.The gene structure,physicochemical properties of proteins,chromosome location,gene evolution,phylogenetic relationship,conserved domain and protein three-dimensional structure modeling of members of GTⅠ genes in japonica rice were analyzed by bioinformatics.The results showed that 30 GT Ⅰ family members of japonica rice were distributed on 12 chromosomes of rice and the promoters of GT Ⅰ family members predicted abscisic acid response.The three-dimensional structure of family proteins was conserved and contained 6 β-folds and 7 α-helix.Transcriptome data and Real-time fluorescence quantitative analysis showed that most members of GT Ⅰ gene family responded to high temperature stress at 6 h after high temperature treatment.The expression level of OS-GT29 was continuously high.GT Ⅰ family was functionally similar in Arabidopsis thaliana and rice.

  • LIU Qili, ZHANG Qianqian, XUE Huali, BI Yang, ZONG Yuanyuan, PENG Hui
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    To further analyze the biological characteristics and expression of TrPLD family genes in Trichothecium roseum,four TrPLD genes were obtained by the whole-genome sequencing of Trichothecium roseum,the online tools of SMART,MEGA 7,ProtScale,SOPMA and other software were used to conduct bioinformatics analysis of TrPLD genes and their encoded proteins;RT-qPCR technology was employed to analyze TrPLD genes expression during the pathogen infection muskmelon fruit.Phylogenetic tree analysis showed that TrPLD1 had the closest genetic relationship with Colletotrichum aenigma with a homology of 63.89%;TrPLD2 had 74.57% homology with Purpureocillium lilacinum;TrPLD3 and TrPLD4 had the closest genetic relationships with Fusarium graminearu and Paecilomyces lilacinus,with homology of 65.38% and 55.45%,respectively.Bioinformatics analysis showed that the proteins of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 all had two conserved domain HKD motifs.TrPLD3 contained PX and PH domains,besides the HKD structure.The four proteins belonged to unstable hydrophilic proteins,without signal peptide and transmembrane structure,and belonged to non-secretory proteins.The four proteins contained different numbers of phosphorylation sites,among which,TrPLD3 contained the most phosphorylation sites.Subcellular localization prediction showed that TrPLD1 and TrPLD3 were mainly located in the nucleus,and TrPLD2 and TrPLD4 were mainly located in the plasma membrane.RT-qPCR analysis showed that,during of simulation the pathogen infecting muskmelon fruit,taking the control expression as a control,the gene expressions of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 increased significantly with the infection process,and the expression of TrPLD3 gene was significantly higher than other genes during the whole infection process.In summary,the four TrPLDs have different structures and biological functions,among which TrPLD3 plays an important regulatory role during the process of T.roseum infecting muskmelon fruit.

  • HU Ziyao, LI Xiuqing, DAI Peihong, LEI Jianfeng, LIU Jianfei, ZHAO Yi, DENG Jiahui, LIU Chao, LIU Xiaodong, LI Yue
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    To explore the biological function of cotton cytochrome P450 gene GhP450-94C1 in cotton Verticillium wilt response, and lay a foundation for cotton Verticillium wilt resistance gene mining and disease resistance breeding. A cytochrome P450 gene, GhP450-94C1, was cloned through transcriptome screening. The physicochemical properties of the gene were analyzed by bioinformatics methods. The expression pattern of GhP450-94C1 under Verticillium wilt induction was analyzed by Real-time quantitative polymerase chain reaction (qRT-PCR). Virus-induced gene silencing (VIGS) technology was used to preliminarily explore its biological function in cotton resistance to Verticillium wilt. The main results were as follows: upland cotton cytochrome P450 gene, GhP450-94C1, was obtained by cloning. The open reading frame (ORF) was 1 503 bp, encoding an acidic, hydrophilic and unstable transmembrane protein with 500 amino acids. The molecular formula was C2597H4025N691O725S22 with a molecular weight of 57.23 ku, which was located in the endoplasmic reticulum membrane and contained a P450 domain. There was 86.45% probability of signal peptide; the secondary structure prediction showed that the protein contained 24 α-helixes and 8 β-sheets. This gene responds to Verticillium wilt infection, and after inhibiting its expression, the sensitivity of plants to Verticillium wilt is enhanced. GhP450-94C1 is a positive regulator of cotton resistance to Verticillium wilt.

  • SUN Haili, LIANG Jing, WANG Wenjia, DING Weihua, WANG Nina, LIU Kaiheng, LI Chengwei
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    AtTUF gene encodes E1 subunit of vacuolar H+-ATP enzyme,and is closely related to the material transport across the tonoplast.In order to explore the tissue expression characteristics and molecular regulatory mechanism of AtTUF,and provide a theoretical basis for the utilization of AtTUF gene promoter and the in-depth study of the regulation characteristics of AtTUF gene expression,the promoter of AtTUF (pAtTUF)was cloned by PCR amplification with Columbia wild-type Arabidopsis as material,the cis-acting elements were predicted through PlantCARE and PLACE,the plant expression vector pBI121-pAtTUF,in which the galactosidase gene(GUS)was driven by pAtTUF,was constructed and transformed into wild-type Arabidopsis to obtain transgenic plants.Through GUS staining,the tissue expression pattern of pAtTUF and its response to hormone and stress were analyzed,and the expression of AtTUF in different tissues was quantitatively analyzed.The results showed that 2 000 bp promoter sequence upstream of the open reading frame of AtTUF gene was successfully cloned.Sequence analysis results showed that pAtTUF contained several cis-acting elements in response to light,abscisic acid(ABA),auxin,methyl jasmonate(MeJA)and drought.The staining results of transgenic lines showed that GUS driven by pAtTUF mainly expressed in the leaves and roots of Arabidopsis seedlings,and in rosette leaves,stems,cauline leaves,sepals,petals,anthers,stigmas,young siliques and immature seeds of adult plants.However, no GUS expression was detected in the hypocotyl at seedling stage,neither in the lateral branches of rosette leaves,mature pods or seeds at adult plant stage.GUS expression intensity was inhibited by ABA,indole acetic acid(IAA),salt,dark treatment and PEG treatment,which was consistent with its characteristics containing a variety of light regulatory elements,hormone regulatory elements and abiotic stress response elements.The Real-time quantitative RT-PCR results showed that AtTUF expressed in roots,stems,leaves,flowers,siliques and seeds,especially higher in siliques at the developmental stage.AtTUF also expressed higher in flowers and dry seeds,but lowest in roots.In conclusion,AtTUF gene plays an important role in the growth and development of plant organs(especially in the reproductive system)and in the response to ABA,IAA,salt,light and drought signals.

  • SONG Hui, GU Binquan, ZHANG Xiangqin
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    To investigate the genetic polymorphism of the inbred lines and hybrids derived from Cucurbita moschata (MO)×Cucurbita maxima (MA),the genetic diversity of 53 pumpkins,including commercial varieties,core parent materials and inter-specific hybrids offspring,were investigated by thirty morphological traits and twenty SSR markers.The results showed that the core parent materials and offspring of inter-specific hybrids had more polymorphism in plant type,growth vigor,fruit skin color and shape than commercial varieties.Twenty SSR primers were screened and showed 90% of polymorphism ratio.Based on the principal components,the cluster results of morphological and SSR markers were consistent with each other very well.The two methods and three times cluster results showed that the commercial varieties and core parent materials of Cucurbita maxima were clustered into one group,firstly.This mean the commercial varieties and core parent materials of Cucurbita maxima had relatively close genetic distance and shared the severely homogenized genetic background.It's very hard to produce the new and different varieties in this situation.Secondly,the offspring of inter-specific hybrids derived from MO×MA were clustered into one group,which was distinguished from commercial varieties and core parent materials of Cucurbita maxima and the genotypes of Cucurbita moschata.The offspring of inter-specific hybrids had relatively long genetic distance and more polymorphism than commercial varieties.It's possible to derive the new and different pumpkin varieties by crossing with the genotypes having the heterogeneous genes produced from interspecies hybrids.Meanwhile,the 6 principal component factors derived from principal component analysis would be used to simplify the edible pumpkin survey indicators.

  • CHEN Xusheng, ZHAO Liang, DI Jiachun
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    In order to analyze a transgenic event of an upland cotton germplasm strain with low expression of a Bt gene,specific primers were used to identify the Bt gene.The PCR amplification result showed a fragment length of the Bt gene was 310 bp,which conformed characteristic primer fragment length to Bt gene of the United States.Using the germplasm strain to cross with island cotton Shengli No.1 showed that the Bt gene was dominant in F1,and Bt-harboring plants to non-harboring plants displayed a 3∶1 segregation ratio in F2 population.The result proved that the Bt gene was a quality trait controlled by a pair of dominant gene,which meant the foreign Bt gene was inserted into upland cotton as a locus.Furthermore,F2 population and SSR markers were used in the gene mapping.The result revealed that the Bt gene was mapped on the 10th chromosome of cotton,and 14 pairs of primers were linked to the target gene,such as NAU5166,NAU3574,NAU456,BNL256,cgr6745,cgr5406,cgr6546,NAU7110,HAU3201,BNL1665,dPL0468,NAU5316,BNL2960,NAU3122.The Bt gene was located between the molecular markers NAU7110 and HAU3201,and its genetic distance was 0.9,4.4 cM,respectively.

  • YUE Dong, YU Zhuo, YU Xiaoxia, LI Jiaqi, LI Jingwei, YANG Dongsheng, ZHANG Hailong, LI Zhicong
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    In order to clarify the genetic differences of the six new-colored potato strains NNCS-1,NNCS-5,NNCS-6,NNCS-7,NNCS-33 and NNCS-37 at the cytogenetic and DNA molecular level,this experiment used material MIN-021 as control,and the regular staining and slides preparing and SSR marker technology were used to analyze the pollen fertility,chromosome configuration at meiosis metaphase Ⅰ of pollen mother cells and SSR polymorphism of the above six varieties.The results shaved that the pollen fertility of the six new-colored potato strains were 44.00%,66.78%,74.44%,60.70%,78.10% and 80.22%,respectively.And the chromosome pairing configurations of these test materials were 8.45Ⅰ+6.09Ⅱ+7.35Ⅲ+1.33Ⅳ,6.91Ⅰ+7.13Ⅱ+5.73Ⅲ+2.41Ⅳ,5.19Ⅰ+9.16Ⅱ+4.51Ⅲ+2.74Ⅳ,6.55Ⅰ+8.35Ⅱ+4.97Ⅲ+2.46Ⅳ,4.02Ⅰ+10.15Ⅱ+3.40Ⅲ+3.37Ⅳ and 3.31Ⅰ+10.42Ⅱ+2.99Ⅲ+3.72Ⅳ,respectively,indicating that there were certain differences among the strains at the cytological level.Ten suitable SSR primers were selected to amplify the genomic DNA of each new strain and control material,and 151 SSR polymorphic sites were obtained,accounting for 86.78% of the total number of polymorphic loci.The genetic differences among the materials were revealed.In addition,a SSR fingerprint was amplified with the selected specific primers C42,which could identify six new-colored potato strains and control.Based on the average genetic distance(GD=0.62),the seven materials were divided into three categories: the new strain NNCS-37,NNCS-6,NNCS-7,NNCS-5 and the control(MIN-021)belong to one categorie.The new strains NNCS-33 and NNCS-1 were separately classified as a categorie.

  • WANG Xintao, YANG Qing, LI Baoye, DAI Ziju, HAO Junjie
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    Leaf length and leaf area are two important agronomic traits,in order to study the genetic mechanism of leaf length and area of three leaves near the ear in maize,a recombinant inbred line(RIL)population(241 lines)derived from Zheng 58 and D863F was used for QTL mapping analysis under spring sowing and summer sowing in 2018 and 2019,respectively.In RILs population, leaf length and area of three leaves near the ear showed continuous variations with a normal distribution and belonged to typical quantitative trait, and significant correlation was found among the traits, the heritability of the six traits was 88.04%, 88.45%, 87.86%, 85.04%, 85.27% and 85.73%, respectively.In total,23 QTLs were detected in two years.The number of QTLs for leaf length and leaf area were 14 and 9,respectively.These QTLs with phenotypic variance explained(PVE)ranging from 5.84% to 17.81% were detected on chromosome 1,2,4,5,6 and 8.Among them,only three QTLs(qFirLL1-2,qFirLL5-1 and qSecLL1-2)for leaf length and two QTLs(qFirLA2-1 and qSecLA2-1)for leaf area were identified in both environments(spring and summer sowing),the remaining QTLs can only be detected separately in spring sowing or summer sowing environments.The major QTLs controlling six traits were detected on chromosome 2(bnlg1316—bnlg1141)and 5(umc1591—umc2298),which explained phenotypic variation ranging from 6.42% to 17.81%;These two marker intervals are important for regulating the length and area of three leaves near the ear, and might contain key genes regulating leaf traits.

  • LIANG Junchao, ZHAO Yunyan, WANG Zhiqi, SUN Jian, YAN Tingxian, YAN Xiaowen, RAO Yueliang, ZHOU Hongying, LE Meiwang
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    To elucidate the genetic mechanism and identify quantitative trait loci(QTL)for Aluminum(Al)tolerance in sesame.We investigated the response of Al stress using sesame landraces Jinhuangma(JHM)and Zhushanbai(ZSB)with different concentrations of Al3+ and confirmed a suitable Al3+ concentration for stress.Three traits including relative root length(RRL),relative shoot length(RSL)and relative fresh seedling weight(RSW)were evaluated in a recombinant inbred line(RIL)population with 180 lines that derived from two parents that contrasts for Al tolerance(ZSB and JHM).Quantitative trait loci(QTL)mapping were conducted by using a high resolution genetic map containing 1 354 bin markers through composition interval mapping(CIM)method.Significant positive correlations were observed among the three traits.A total of 7 QTLs were detected on 6 chromosomes(Chr2,Chr4,Chr6,Chr10,Chr11 and Chr13),including 3 RRL related QTLs,2 RSL related QTLs and 2 RSW associated QTLs.Among them,two QTLs associated with RRL and RSW were mapped to the overlapped interval on chromosome 2.Putative candidate gene analysis revealed that 15 genes may be related to Al tolerance.These genes mainly involved in metal transport and detoxification,transmembrane binding of metal ions,GDSL-motif lipase,peroxisomal catalase and metabolism and detoxification of toxic substances in organisms.

  • Tillage & Cultivation·Physiology & Biochemistry

  • LIU Yanan, LU Zhanyuan, SUN Fengcheng, ZHANG Xiangqian, ZHANG Jiaqian, BAO Eerdunga, FENG Ye, YU Yang, QIAN Defang
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    In order to explore the effects of planting density on grain moisture content,dehydration rate and mechanical harvesting quality of different maize varieties,to clarify the dehydration law and mechanical harvesting loss characteristics of maize grains under different densities at harvesting stage,and to provide theoretical basis for determining the appropriate mechanical grain harvesting time under dense planting mode of spring maize.Five maize varieties Zhengdan 958(ZD958),Xianyu 335(XY335),Lishou 1(LS1),Zhongnong 222(ZN222)and Zhongnong 239(ZN239)were used as experimental materials,and planting densities of 60 000,75 000 and 90 000 plants/ha were set.The effects of different density treatments on grain moisture content,dehydration rate and harvesting quality of spring maize were analyzed.The results showed that planting density significantly affected the grain water content at mature stage,and the grain water content of 90 000 plants/ha was significantly lower than that of 75 000,60 000 plants/ha,while there was no significant difference between the two low density treatments.There was a great difference in water content among different varieties,and the water content from high to low in two consecutive years were ZD958>LS1>ZN222>XY335>ZN239.Planting density had no significant effect on dehydration rate,but variety had a significant effect on dehydration rate.With the increase of planting density,the water content and fragmentation rate of seed decreased.The results showed that ZD958 and XY335 were not suitable for local mechanical harvesting,while LS1,ZN239 and ZN222 were suitable for local mechanical harvesting.The optimum density is from 75 000 to 90 000 plants/ha.

  • KUANG Wei, WEI Zheng, DAI Li, ZHAO Yang, LIANG Yugang, LUO Xianfu, ZHANG Yuzhu, FANG Baohua
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    In order to solve the problem of the reduction of the migration of young rural labor force and the aging of the population,the enthusiasm for food crop production has been greatly reduced,and it is urgent to explore rice planting methods with less labor input and matching high-yield varieties.Through field trials from 2017 to 2019,two common and easy-to-promote planting methods were compared,and 14 early rice varieties and 12 late rice varieties were screened for easy purchase.The planting methods were simulated machine transplanting and machine direct seeding.Effects of different planting methods on the growth period,yield and dry matter weight of different early and late rice varieties.The results showed that direct seeding could shorten the growth period compared with transplanting.The average growth period of early rice direct seeding was shortened by 7 d compared with transplanting,and the average growth period of late rice was shortened by 8 d.The growth period of direct seeding had a smaller fluctuation range and more stable performance than transplanting.The annual average yield was significantly higher than that of transplanting,which was 29.71% higher in 2017,12.37% higher in 2018,and 7.15% higher in 2019,and it was found that the yield and dry matter accumulation at different periods were affected by the variety,the planting method and year had a very significant effect;through the linear regression covariance test,there was a positive correlation between the yield and the dry matter accumulation,which showed that the yield increases with the increase of the dry matter accumulation,and the coefficient of determination of the linear regression of the direct seeding method(R2)were higher than the transplanting method.A comprehensive comparison showed that the direct seeding method performs better in both the early and late rice planting methods,and the stable and high-yield(high-yield performance in the field in three years)that is matched with the direct seeding method is screened.The early rice variety Zhuliangyou 829(the yield fluctuation range of 6.02—10.90 t/ha),Yuliangyou 4156(6.49—10.22 t/ha),and stable and high-yielding late rice variety Wuyou 308(10.43—12.65 t/ha),the selected early and late rice varieties had moderate growth periods and can be effectively connected achieve high-yield planting of early and late rice.

  • Resources & Environment·Plant Protection

  • SHAO Yun, MA Yueying, HOU Meng, YANG Junhua, MA Guanqun
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    Under the double cropping pattern in the North China Plain,based on the conventional application level of phosphorus,a phosphorus-deprived level without phosphorus fertilizer was adopted at the same time for three previous crops,corn,soybean and peanut,and then the soil nutrient concentration after previous crop harvesting and the grain yield and nutrient accumulation of winter wheat,the subsequent crop,were examined,to provide a proposal for crops planting in the North China Plain.The results showed that,for the soil nutrients after the previous crop harvesting,the soil total phosphorus concentration was higher in the previous soybean treatment without phosphorus fertilizer,and the concentration in peanut previous treatment was similar to that under conventional fertilizers;the soil available phosphorus content was higher overall in the previous peanut treatment;the soil nitrate and ammonium nitrogen concentrations were both highest in the previous soybean treatment.For post-crop wheat,wheat thousand grains weight,yield,seed N and P accumulation and N fertilizer bias productivity were all higher in the previous peanut treatment without phosphorus fertilizer.The previous peanut treatment increased by 0.60%,6.19%,15.46%,18.11% and 6.21%,respectively,compared to the previous maize treatment,and increased by 2.18%,7.30%,17.66%,13.40% and 7.30%,respectively,compared to the previous soybean treatment.In summary,in order to ensure soil nutrient balance,peanut was selected as a suitable previous crop in summer before winter wheat as a better model for crop mix in the southern two maturity zones of the North China Plain at low phosphorus levels.

  • SHI Lihong, SUN Mei, TANG Haiming, LONG Zedong, SUN Geng, WEN Li, LI Chao, LUO Zunchang
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    In order to explore the effects of different long-term (36 a)fertilizer treatments on characteristics of soil acid buffering capacity at different soil layers (0—10 cm and 10—20 cm)under the double-cropping rice field in Southern of China,four different fertilizer treatments were set up:without any fertilizer input as a control(CK),chemical fertilizer alone (MF),rice straw and chemical fertilizer (RF),and 30% organic manure and 70% chemical fertilizer (OM).Soil acid neutralizing capacity(ANC),acid buffering capacity(ABC),instant acid buffering capacity(IAC),ammonium nitrogen($\text{NH}_{4}^{+}$-N),nitrate nitrogen($\text{NO}_{3}^{-}$-N)contents,and its relationship between soil acid buffering capacity and soil physicochemical properties were analysis.This result indicated that soil ANC,ABC and IAC at soil 0—10 cm and 10—20 cm layer in the double-cropping rice field with MF,RF and OM treatments were increased.The order of soil ANC,ABC and IAC at soil 0—10 cm and 10—20 cm layer in paddy field with all fertilizer treatments showed OM>RF>MF>CK.Compared with CK treatment,soil ANC and ABC at 0—10 cm and 10—20 cm layer in paddy field with OM treatment increased by 36.78%,33.18% and 18.67%,17.84%,respectively.Soil IAC at 0—10 cm and 10—20 cm layer in paddy field with OM treatment increased by 15.22% and 14.02%,compared with CK treatment,respectively.This result indicated that soil $\text{NH}_{4}^{+}$-N content at 0—10 cm and 10—20 cm layer in paddy field with MF treatment were significantly higher than that of RF,OM and CK treatments.Soil $\text{NH}_{4}^{+}$-N content at 0—10 cm and 10—20 cm layer in paddy field with MF treatment increased by 48.15% and 51.09%,compared with CK treatment,respectively.This result showed that soil $\text{NO}_{3}^{-}$-N content at 0—10 cm and 10—20 cm layer in paddy field with OM treatment were significantly higher than that of MF,RF and CK treatments.Soil $\text{NO}_{3}^{-}$-N content at 0—10 cm and 10—20 cm layer in paddy field with OM treatment increased by 204.73% and 161.94%,compared with CK treatment,respectively.Soil ANC,ABC,IAC,$\text{NH}_{4}^{+}$-N and $\text{NO}_{3}^{-}$-N contents at 0—10 cm layer were obvious higher than that of 10—20 cm layer in paddy field under the same fertilizer treatment condition.It had significantly positive correlation between soil ANC,ABC,IAC and soil organic carbon,total nitrogen contents,electrical conductivity in paddy field.Meanwhile,there had extremely significantly positive correlation between soil ANC,ABC,IAC and soil pH,cation exchange capacity in paddy field.As a result,it was effective practices for increasing soil acid buffering capacity at plough layer under the double-cropping rice field in Southern of China by combined application of rice straw or 30% organic manure with chemical fertilizer managements.

  • LÜ Lihua, YAO Haipo, CAO Zhimin, ZHANG Jingting, YAO Yanrong, JIA Xiuling
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    It explored the ratio of organic fertilizer replacing nitrogen fertilizer in the piedmont plain of Hebei Province,in order to provide a basis for reducing the amount and increasing the efficiency of nitrogen in wheat in this area.Field experiments were carried out in Boyuan farm in Yongnian,Hebei Province for two consecutive years,and five organic and inorganic fertilizer combination treatments were set up.The results showed that organic fertilizer instead of 20% and 40% chemical fertilizer could significantly improve the number of grains per spike and yield.Compared with the high nitrogen and saving nitrogen treatment of single chemical fertilizer application,the yield increased by more than 4.0%,and the number of grains per spike increased by 3.6—5.6.Most of the grain quality indexes for organic fertilizer instead of 20% and 40% chemical fertilizer treatment,and saving nitrogen treatment were better,and the stabilization time increased by 2.2—2.7 min,the tensile area increased by 10.5—17.5 cm2,and the maximum tensile resistance increased by 28.0—75.5 EU.Various nitrogen efficiency indicators of treatment for organic fertilizer instead of 20% were higher.The nitrogen fertilizer efficiency,nitrogen utilization efficiency,and nitrogen harvest index increased 109.3%,9.3% and 11.3% respectively compared with high nitrogen treatment,and 6.9%,8.5% and 8.3% respectively compared with the saving nitrogen treatment.When organic fertilizer replaced chemical fertilizer in different proportions,nitrate nitrogen in 0—20 cm soil appeared "surface accumulation",and the content of nitrate nitrogen increased,which was more than 38.5% higher than that of the saving nitrogen treatment.The nitrate nitrogen in 20—40 cm soil was significantly higher for the saving nitrogen treatment and the high nitrogen application treatment.Organic fertilizer instead of 20% nitrogen fertilizer treatment had the best yield and grain quality,significantly improve the nitrate nitrogen content in 0—40 cm soil,improve the nitrogen absorption and utilization of wheat,and finally obtain higher environmental benefits.

  • LU Yan, GAO Jian, KANG Wenqin, YUAN Xili, JING Lan
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    To explore the abundance of Gibberella zeae and the composition and diversity of fungal communities in spring wheat belt of the Northeast, bulk soil samples were collected under four crop rotation patterns, such as wheat-wheat-potato (T), wheat-wheat-silybum marianum (MT), wheat-wheat-oilseed rape (R), wheat-wheat-sugarbeet (S), and wheat for three years (W) was used as control. The Illumina MiSeq was used to sequence the ITS amplicons of the strains. The data showed that the number of fungal OTUs in W, T, MT, R and S was 389, 362, 390, 471 and 438, respectively. In the R and S rotation pattern, Chao1 were increased 11.08% and 8.59% respectively, compared with wheat continuous cropping, indicating that the rotation of silybum marianum, oilseed rape and sugarbeet with wheat increased the abundance of fungi genus. Among the five planting patterns, Shannon and Simpson in MT were the highest, revealed that the diversity of fungal communities in MT was more enriched. The similarity cluster analysis of fungal community structure showed that the four rotation patterns clustered into one branch. Under the four cropping rotation patterns, the abundance of basidiomycota increased while that of Zygomycota decreased. We also found that the relative abundance of Gibberella zeae decreased by 16.67%, 50.00% and 83.33% in R, S and MT, respectively, while the abundance of friendly genus were remarkably increased. However, the relative abundance of Gibberella zeae was increased significantly in the T pattern. Based on the above research results and the local planting structure, it is suggested to plant silybum marianum, sugarbeet and oilseed rape rotation with wheat to reduce the hazard of wheat scab.

  • BEN Haiyan, HAO Yongjuan, HUO Jianfei, YAO Yurong, GAO Wei, WANG Wanli
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    Strawberry powdery mildew caused by Sphaerotheca aphanis is one of the strawberry diseases with large incidence area and high incidence frequency in the protected cultivation areas, which usually occurs from seedling stage to fruiting stage. Therefore, the establishment of a rapid and efficient detection method of S. aphanis is quite important for the diagnosis and control of strawberry powdery mildew.A set of LAMP amplification primers including F3/B3、FIP/BIP and LF/LB were designed for the conserved sequence of ITS gene of Sphaerotheca aphanis as the target gene. Through the double judgment method of Real-time fluorescence curve and fluorescence color change, the main factors in the LAMP reaction system including the final concentrations of dNTPs, MgSO4, BstDNA polymerase and betaine and amplification temperature were selected and optimized to increased etections ensitivity and specificity. Finally, the effects of field samples detection by optimized LAMP were evaluated.The results showed that the LAMP detection system of strawberry powdery mildew was established successfully. The optimized reaction conditions included dNTPs 1.6 mmol/L, MgSO4 8 mmol/L, BstDNA polymerase 320 U/moL and betaine 1.2 mmol/L and the amplification temperature was 65 ℃. Under such conditions, the minimum detection sensitivity was 3.2×10-4 ng/μL within 60 min, and the efficiency of which was 100 times higher than the result of PCR amplification.The visual LAMP method could effectively detect the samples with no obvious symptoms infected by S. aphanis in the early stage. This method has the advantages of short detection time, direct observation with eyes, low requirements for operators and low detection cost. It has important guiding significance for the early diagnosis, monitoring, early prevention and determination of the best control time of strawberry powdery mildew.

  • ZENG Yanming, HU Guang, JIA Pei, TANG Ye, WANG Bingting, WU Pan, LU Chengzhe, CHEN Aimin, PENG Qingzhong, WU Jiahe
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    To eradicate Verticillium wilt of cotton,identification of resistant genes and breeding of resistant cotton varieties are the best methods at present.We used MEGA 5.2 and other related software to construct phylogenetic trees of the proteins encoded by WRKY7 genes in Gossypium hirsutum and Arabidopsis thaliana and Oryza sativa.The induction and response of GhWRKY7 gene to Verticillium dahliae and its mechanism were elucidate by subcellular localization,virus-induced gene silencing,qPCR and GUS reporting system analysis.The results showed that GhWRKY7 and AtWKRY7 were highly homologous and belonged to Group Ⅱ.GhWRKY7 was located in plant nucleus,and the relative expression of GhWRKY7 in cotton leaves and root organs was significantly higher than that in stems.GhWRKY7 gene expression was significantly upregulated after 24 h of inoculation.The GhWRKY7-silenced plants showed higher susceptibility to Verticillium dahliae infection compared to the control(Expression TRV empty vector plants),suggesting that GhWRKY7 gene positively regulated cotton disease resistance.Compared to the control,the expression levels of disease-resistance related genes,including GhPR1,GhPR3,GhPR4,GhPR5,GhPDF1.2,GhPAL1 and GhCYP71B36 in GhWRKY7-silenced plants significantly decreased after inoculation,indicating that GhWRKY7 improved the disease resistance of the plants due to increased the expression levels of disease-resistance related genes.Transient expression analysis of GhWRKY7 gene by constructing GUS reporter vector in tobacco cells revealed that GhCYP71B36 gene could specifically bound to cis-element of GhCYP71B36 promoter and transcriptionally activated downstream GhCYP71B36 expression,thus improved the disease resistance of cotton.In conclusion,GhWRKY7,as a transcription factor that positively regulates Verticillium wilt resistance in cotton,is involved in the expression of downstream disease-resistance related genes such as Camalexin synthesis,thereby improving plant disease resistance.Therefore,GhWRKY7 can be used as a candidate gene for cotton resistance breeding and provide security for cotton production.

  • Animal Husbandry·Fisheries·Veterinarian

  • SHAO Ying, HUANG Yan, YANG Yan, GONG Liufei, SONG Xiangjun, TU Jian, QI Kezong
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    It aimed to develop a polyclonal antibody to Avian leukosis virus (ALV)and to establish a simple and accurate method for the detection of ALV.pGEX-6p-1-p27 and pET-32a-p27 protein vectors were constructed by amplifying the target gene of ALV p27.The proteins were immunized in BALB/c mice and New Zealand white rabbits,and mouse-derived polyclonal antibodies and rabbit-derived polyclonal antibodies were prepared.The mouse-derived polyclonal antibody coupled fluorescent microspheres were prepared,and the mouse-derived polyclonal antibody IgG was sprayed onto the sample pads using a three-dimensional spray point platform;the purified rabbit-derived polyclonal antibody IgG and goat anti-rabbit antibody dilution were scribed onto the NC membrane using a scribing instrument as T and C lines for the assembly of test strips,and a preliminary Point-of-care testing(POCT)immunochromatographic assay was established.The test strips were assembled and a preliminary point-of-care testing(POCT)method was developed for the detection of ALV by fluorescent microsphere immunochromatography.The results showed that the prokaryotic expression vectors for pGEX-6p-1-p27 and pET-32a-p27 were successfully constructed and the expression of p27 recombinant protein was induced.After mixing the purified p27 fusion protein with the adjuvant as an immunogen immune BALB/c mouse and a New Zealand white rabbit,a mouse-derived and rabbit-derived polyclonal antibody of the p27 protein was successfully prepared,and the immunogen response of the multi-antibody was shown to be good by serum potency and Western Blot identification.The optimal pH of the fluorescent microsphere-coupled murine polyclonal antibody was 6.2.POCT strips showed that positive samples for ALV bound well to the fluorescent microsphere-coated murine polyclonal antibody,with higher T/C data than other samples,and that positive samples bound to the fluorescent microsphere-coated polyclonal antibody showed fluorescent bands in the C line(quality control line)and T line(detection line)under UV light,which the results were consistent with clinical diagnostic results.It can be concluded that the POCT fluorescent microsphere immunochromatographic detection method for ALV established provides a simpler method for the detection of Avian leukosis virus and facilitates clinical detection for primary veterinarians.

  • XIANG Ya, ZHONG Jincheng, WU Zhijuan, CHAI Zhixin
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    In order to explore the expression patterns of TNNI1 and TNNI3 in the striated muscle of Maiwa yak,this study selectd the gluteal muscle and myocardial tissue of Maiwa yak of different ages,morphological changes observed.The expression trend of TNNI1 gene in gluteal muscle tissue of Maiwa yak at different ages and its association with muscle fiber traits were carried out,the mRNA,protein differential expression and protein localization of TNNI1 and TNNI3 in myocardium were analyzed.The results showed that the muscle fiber diameter and density had certain characteristic changes with the increase of yak age.The muscle fiber diameter increased significantly with the increase of age,while the muscle fiber density had the opposite change.The mRNA expression of TNNI1 gene in gluteal muscle tissue of adult yaks was significantly higher than calves and weaned calves. TNNI1 mRNA expression in gluteus muscle was significantly positively correlated with muscle fiber diameter,and significantly negatively correlated with muscle fiber density.The mRNA expression level of TNNI1 in myocardium decreased with the increase of yak age,while the expression level of TNNI3 showed a trend of first increased and then decreased,and the mRNA expression level of TNNI1 was the highest in calf stage,while TNNI3 showed an upward trend with the increase of age.The expression levels of TNNI1 and TNNI3 proteins in myocardium decreased with the increase of yak age.The expression levels of TNNI1 and TNNI3 proteins in myocardium decreased with the increase of yak age.The TNNI3 mRNA expression decreased with the increase of yak age,which was consistent with the trend of protein expression.It provided basic data for exploring the regulation mechanism of TNNI1 and TNNI3 on yak muscle growth and development.TNNI1 and TNNI3 have significant regulatory effects on the growth and development of the muscle tissue of Maiwa yak at different ages,and the expression levels in the muscle tissue of yak at different ages are different.

  • YANG Manzhen, MIN Xingyu, YANG Luyu, YU Hailing, HU Yulei, ZHU Yanjin, PAN Bangting, LI Jian, XIONG Xianrong
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    The aim of this study was to clone the kinesin family member 2A gene(KIF2A),and explore the expression pattern of KIF2A gene in different tissues of yak,as well as the spatially and temporally expressed during the follicular development and oocyte meiotic maturation.The heart,liver,spleen,lung,kidney stomach,uterus and ovary tissues(n=5)were collected from 3—4 years old healthy female yak after slaughter.Yak KIF2A gene was amplified by RT-PCR with yak ovarian cDNA as template,and its protein were analyzed by bioinformatics software such as MAGA 7.0 and SWISS-MODEL etc.Quantitative Real-time PCR(RT-qPCR)was used to detect the expression level of KIF2A in yak various tissues and immunohistochemistry was used to analyze the expression and localization of KIF2A in follicles with different diameters.According to the diameter,the follicle were divided into three groups: small(diameter 1.0—2.9 mm),medium(diameter 3.0—5.9 mm),and large(diameter 6.0—9.0 mm)follicle.The expression characteristics of KIF2A gene in granulosa cells and oocyte meiosis in follicles were analyzed by RT-qPCR.The results showed that sequence length of KIF2A gene was 1 964 bp,and the CDS was 1 530 bp,which encoding 509 amino acids.KIF2A protein was negatively charged and belonged to hydrophobic protein.KIF2A gene was relatively conservative and widely expressed in various tissues of yaks, and KIF2A protein was mainly located in granulosa cells, its mRNA expression in follicular granulosa cells with different diameter and size increased with the growth and development of follicles. During the meiotic maturation of yak oocytes,the expression of KIF2A showed a sequential feature,and the G Ⅴ phase was significantly higher than that of M Ⅰ and M Ⅱ phase.The results showed that KIF2A might be involved in the regulation of follicular and oocyte maturation development in yaks,and the results of this study provided basic data for the functional study of KIF2A gene in yak follicular development.

  • LI Jie, HUANG Xiaoyu, SHANG Xuefeng, YANG Jiaojiao, ZHANG Juanli, XIE Kaihui, ZHANG Yali, YAN Zunqiang, WANG Pengfei, GAO Xiaoli, YANG Qiaoli, MA Yanping, GUN Shuangbao
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    The purpose of this study was to analyze the differential expression of FABP2 in different tissues of DLY fattening pigs fed different proportions of Zanthoxylum seeds instead of some corn,and to find the key gene for intramuscular fat deposition;at the same time,the sequence of the coding region of FABP2 in DLY fattening pigs was cloned and analyzed by bioinformatics methods.A total of 288,90-day-old Duroc Landrace Yorkshire fattening pigs(33 kg)were randomly divided into 4 groups with 4 replicates and 18 pigs in each replicate;the control group was fed the basal diet and the test group was fed Zanthoxylum seeds instead of corn in the diet at 2.5%(test group Ⅰ),5.0%(test group Ⅱ)and 7.5%(test group Ⅲ),respectively,for 7 d in the pretest and 100 d in the test period.PCR was used to detect the relative expression of FABP2 in different tissues of the test groups,and the coding region of FABP2 in DLY fattening pigs was also cloned,and bioinformatics analysis software was used to predict the physicochemical properties,structural domains and phosphorylation kinase sites of FABP2 protein.The results showed that FABP2 was differentially expressed in all tissues,with significantly higher expression in jejunum and liver.Compared with the control group,FABP2 was significantly down-regulated in heart,lung,kidney,duodenum,jejunum and cecum tissues in each test group;FABP2 was significantly up-regulated in spleen and ileum tissues in test group Ⅱ and test group Ⅲ;FABP2 was significantly up-regulated in liver,colon,rectum and dorsal muscle tissues in test group Ⅰ,indicating that the addition of Zanthoxylum seeds in the diet had a significant effect on the expression profile of FABP2 gene in DLY fattening pigs.In this experiment,the full length(399 bp)of the coding region of FABP2 was successfully cloned and identified,encoding 132 amino acids,with two missense mutations;FABP2 protein was an acidic stable non-secretory protein with a secondary structure consisting mainly of extended chains and random coils,containing a Lipocalin-FABP2 superfamily conserved structural domain and three disordered regions.

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