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    01 November 2021, Volume 54 Issue 21
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Genome-Wide Association Analysis for Yield and Nitrogen Efficiency Related Traits of Wheat at Seedling Stage
    ZHANG PengXia,ZHOU XiuWen,LIANG Xue,GUO Ying,ZHAO Yan,LI SiShen,KONG FanMei
    Scientia Agricultura Sinica. 2021, 54(21):  4487-4499.  doi:10.3864/j.issn.0578-1752.2021.21.001
    Abstract ( 700 )   HTML ( 109 )   PDF (4907KB) ( 522 )   Save
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    【Objective】 To identify and locate the molecular markers which were stable and significantly correlated to the traits of biomass and N efficiency under different N nutrition levels will help to provide reference for cloning and characterization of the related genes.【Method】A group of 134 wheat varieties (or lines) were used in a two-years (2013 and 2014) hydroponic experiments, in which three treatments applying normal level N, low level N and high level N were set up. Fourteen traits related to biomass and N efficiency were measured, as the respective average values of each treatment in one year and two years. Genome-wide association analysis using 90K SNP molecular markers was carried out for the tested traits by MLM+K+Q mixed linear model. 【Result】 Compared with normal nitrogen treatments, roots, shoots, and plant nitrogen content and nitrogen accumulation were significantly reduced in low nitrogen treatments, while root biomass and root and plant nitrogen efficiency were significantly increased. In high nitrogen treatments, almost all traits are significantly increased. The heritability of all the tested traits were above 40%. According to genome-wide association analysis on the 9 329 SNPs, a total of 838 molecular marker sites were identified associating with 14 traits significantly (P≤0.001). These markers located on 21 chromosomes, among which 435 (51.91%) molecular marker sites were detected in only one environment, 403 and 8 environment stable sites were identified in at least two or three environments, two environmental stable SNP marker sites were identified in at least four environments. The two stable markers (Kukri_c65481_121 and tplb0025f09_1052) were significantly related to total nitrogen use efficiency of plant (TNUE) and root nitrogen use efficiency (RNUE), respectively. Five multi-trait co-location SNP marker sites which simultaneously associated with at least six traits were located on chromosomes 1A, 1B(2), and 2A(2). Furtherly, candidate gene prediction was conducted in the 214 kb genomic region of 5 SNP sites co-located with 6 traits (biomass and N efficiency traits) and 2 SNP sites associated with multiple environments (4 environments). According to the genome annotation and LD attenuation level, a total of 84 candidate genes were determined. Gene function annotations of these genes were performed using the coding protein types of known cloned nitrogen efficiency genes, candidate gene function annotation information and the use of plant comparative genomics resource library protein sequence homology analysis, 3 candidate genes were initially determined. 【Conclusion】 Different N treatments significantly affected the phenotypic traits of biomass, N efficiency and the expression of related QTLs at seedling stage of wheat. Most SNPs were detected in only one N environment, but there were some locations with relatively strong environmental stability. There was a significant correlation between biomass and N efficiency related traits, and they might be partly controlled by the same QTL/gene. The functions of related candidate genes related to N efficiency and biomass of wheat selected in this paper needed to be further verified.

    Over-expression of ZmIBH1-1 to Improve Drought Resistance in Maize Seedlings
    ZHU FangFang,DONG YaHui,REN ZhenZhen,WANG ZhiYong,SU HuiHui,KU LiXia,CHEN YanHui
    Scientia Agricultura Sinica. 2021, 54(21):  4500-4513.  doi:10.3864/j.issn.0578-1752.2021.21.002
    Abstract ( 632 )   HTML ( 57 )   PDF (2129KB) ( 404 )   Save
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    【Objective】Drought is an important factor that affects the growth and development of maize seriously. Through the mining of genes related to drought resistance in maize, transgene function verification and transcriptome analysis, analyzing the molecular regulation mechanism of key genes in response to drought stress, this paper provides the theoretical basis for drought resistance molecular breeding and genetic improvement. 【Method】In this study, the maize inbred line B104 (wild-type, WT) was used as the background to construct the ZmIBH1-1 overexpression (ZmIBH1-1-OE) transgenic line by Agrobacterium-mediated method. The transgenic plants and lines were identified via screening transgenic plants for glufosinate-ammonium resistance, PCR detection of marker gene and target gene and expression analysis of target gene by qRT-PCR. We used the ZmIBH1-1-OE and WT transgenic lines as materials. Through drought treatment (20% PEG6000), phenotype identification and drought tolerance physiological and biochemical index determination were carried out to verify the drought resistance function of ZmIBH1-1; RNA-Seq was used to identify differentially expressed genes (DEGs) under drought stress at the 4-leaf stage; Combined with DAP-seq (DNA affinity purification sequencing) analysis, it is preliminarily determined that ZmIBH1-1 protein directly regulates downstream target genes related to drought resistance, and IGV (Integrative Genomics Viewer) was used to analyze the position of the ZmIBH1-1 protein binding candidate target gene, and then the Dual-Luciferase assay was used to verify the regulatory relationship between ZmIBH1-1 protein and target genes. 【Result】12 transformation events were obtained by genetic transformation of maize. In the T3 generation, there were 458 plants in which the marker gene Bar and the target gene ZmIBH1-1 were simultaneously detected. The results of qRT-PCR showed that the expression level of ZmIBH1-1 in ZmIBH1-1-OE lines was significantly higher than that of WT and the expression levels of transformation events 3 and 8 were the highest, which were self-crossed to obtain T4 generation for subsequent experiments. Under drought stress, the survival rate, the relative water content, the chlorophyll content, soluble protein content and the physiological and biochemical indicators (superoxide dismutase, peroxidase, catalase activity) of ZmIBH1-1-OE were higher than those of WT significantly, which indicating that the overexpression of ZmIBH1-1 in maize confers higher drought tolerance. The RNA-Seq results showed that there were 1 214 DEGs between WT and ZmIBH1-1-OE lines. Gene Ontology (GO) analysis showed that DEGs were mainly involved in biological processes, cell components and molecular functions, such as photosynthesis, stress response, dehydration response, etc. in biological processes; KEGG enrichment analysis showed that DEGs were mainly involved in the signal transduction of plant hormones, the metabolism and other processes. Combining the significantly DEGs of RNA-Seq and the target genes of ZmIBH1-1 obtained from DAP-seq analysis, it is preliminarily identified 11 candidate target genes related to drought resistance that may be directly regulated by ZmIBH1-1, including 2 calcium signal related genes, 3 cysteine metabolism related genes, 1 bHLH transcription factor, 1 stress response protein, 1 glutathione transferase, 1 redox process protein and 2 ethylene response factor; Integrative genomics viewer showed that ZmIBH1-1 protein could bind to the promoters of the target genes; Subsequent Dual-Luciferase assay further showed that ZmIBH1-1 protein can directly act on 11 candidate target genes, of which, ZmIBH1-1 directly binds to the promoters of ZmCa-M, ZmSYCO, ZmbHLH54, ZmGlu-r1, ZmCLPB3 and ZmP450-99A2 to promote their expression, and directly binds to the promoters of ZmAGD12, ZmCYS, ZmCYSB, ZmERF-107 and ZmEIN3 to repress their expression. In addition, transcription factors such as NAC, WRKY and MYB also differentially expressed between WT and ZmIBH1-1-OE under drought stress. 【Conclusion】The overexpression of ZmIBH1-1 can enhance the drought tolerance of maize; ZmIBH1-1 improves the drought tolerance of maize by directly regulating the expression of genes ZmERF-107 and ZmEIN3 in the ethylene signaling pathway; ZmIBH1-1 enhances the drought tolerance of maize by directly regulating the calcium signal-related genes ZmCa-M and ZmAGD12; ZmIBH1-1 may indirectly regulate NAC, WRKY, MYB and other transcription factors in response to drought stress.

    Optimization of Cotton Mesophyll Protoplast Transient Expression System
    LI Qing,YU HaiPeng,ZHANG ZiHao,SUN ZhengWen,ZHANG Yan,ZHANG DongMei,WANG XingFen,MA ZhiYing,YAN YuanYuan
    Scientia Agricultura Sinica. 2021, 54(21):  4514-4524.  doi:10.3864/j.issn.0578-1752.2021.21.003
    Abstract ( 669 )   HTML ( 33 )   PDF (4819KB) ( 452 )   Save
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    【Objective】Cells of true leaves can well mimic the plant endogenous situation. It is an efficient way for expediting cotton functional study to establish an effective transient expression system using cotton protoplasts obtained from true leaves.【Method】The enzyme combination of cellulose and macerozyme were used to isolate protoplasts from true leaves of Gossypium hirsutum L. acc. TM-1. The effects of osmotic pressure, components of digestion buffer and digestion time on protoplast yield were studied and the validity of protoplasts were compared under different mannitol concentration and digestion time. To improve the transformation efficiency of cotton protoplast, the effects of mannitol and PEG concentration and buffers for protoplast culture were subsequently studied. In order to verify the optimized transient expression system, the vector 35S:LTP-GFP was constructed and transformed into protoplasts of Arabidopsis and cotton and tobacco epidermal cells followed by observation of fusion protein localization.【Result】High concentration of CaCl2 in the digestion buffer significantly inhibited the isolation of protoplast from cotton true leaves, which was opposite of that using cotyledon. 10 mmol·L -1 CaCl2 was employable for digestion buffer to isolate cotton protoplasts from true leaves. Mannitol concentration significantly affected protoplast yield that peaked under mannitol concentration of 0.5 mol·L-1, and protoplast validity decreased moiety under 0.4 mol·L-1 mannitol, suggesting that 0.5 mol·L-1 mannitol was most suitable to maintain the osmotic pressure of cotton protoplasts. Cotton protoplasts displayed suitable size when isolated from newly flattened true leaves, while protoplast enlarged and yield decreased when produced from young leaves flattened 5 days. The protoplasts dissociate slowly until being digested 9 h when the yield reached the peak. The transformation efficiency was greatly improved under isotonic condition of 40% PEG buffer. While hypotonic condition that is commonly applied to facilitate transformation was against the entrance of exogenous DNA into cotton protoplasts. After transformation, the protoplast ruptured abundantly in WI buffer,whereas the shape maintained well in W5 buffer adding 0.5 mol·L-1 mannitol. The transformation efficiency was improved to 90% using the optimized transient expression system. The subcellular location analysis results showed consistent GFP signal in protoplasts of cotton and Arabidopsis true leaf and epidermal cells of tobacco leaf.【Conclusion】Our study has optimized the cotton mesophyll protoplast transient expression system, which could produce 8.10×10 6·mL-1 fine protoplasts with validity above 95% and transformation efficiency reached to 90%. This system is applicable for analysis of subcellular location, protein interaction and research on metabolism and regulation network.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Determination of Suitable Band Width for Estimating Rice Nitrogen Nutrition Index Based on Leaf Reflectance Spectra
    XU HaoCong, YAO Bo, WANG Quan, CHEN TingTing, ZHU TieZhong, HE HaiBing, KE Jian, YOU CuiCui, WU XiaoWen, GUO ShuangShuang, WU LiQuan
    Scientia Agricultura Sinica. 2021, 54(21):  4525-4538.  doi:10.3864/j.issn.0578-1752.2021.21.004
    Abstract ( 332 )   HTML ( 38 )   PDF (2699KB) ( 327 )   Save
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    【Objective】The research aimed to analyze the relationship between rice (Oryza sativa L.) nitrogen nutrition index (NNI) and leaf spectral reflectance characteristics of leaf on different positions, so as to provide an effective method for nondestructive and timely evaluation of NNI in rice.【Method】Field experiments were conducted with different N application rates and rice cultivars across two growing seasons during 2018-2019, and the leaf hyperspectral reflectance of 350-2 500 nm of leaf on different positions and the plant NNI were measured during key fertility growth stages to construct a variety of spectral index model for rice NNI monitoring.【Result】The results indicated that green band (540 nm) at leaf level was the sensitive band for estimating NNI, and narrow band ratio index SR (R900, R540) composed of near infrared band and green band could be used to retrieve NNI of rice. However, the prediction accuracy of narrow band ratio index and rice NNI of leaf on different positions were different. In terms of prediction accuracy, the best single leaf position was the third leaf (L3) from the top (R2=0.731, RMSE=0.130, RE=11.6%), the second leaf (L2) from the top followed (R 2=0.707, RMSE=0.136, RE=12.2%), and the top one (L1) was the worst (R 2=0.443, RMSE=0.187, RE=14.7%). The averaged spectra of L2 and L3 (L23) was the optimum leaf spectra combination, which contributed to improving the predictability to NNI(R 2=0.740, RMSE=0.128, RE=11.5%). The samples were resampled at 50 nm and 10 nm in the near infrared region (900 nm) and green region (540 nm) respectively, and the accuracy of the wide band ratio index SR (AR(900±50), AR(540±10)) was not significantly lower than that of SR (R900, R540). The model accuracy and prediction accuracy of the two models were basically the same at L23. When the NNI of rice was less than 1, there was a significant positive linear correlation with the yield, and then it tended to be stable. 【Conclusion】The results showed that the reflectance spectra of L2 and L3 leaves were sensitive for monitoring NNI for rice, and L23 could improve the prediction accuracy of the model. Multiple band ratio indices SR (R900, R540) and SR (AR(900±50), AR(540±10)) based on leaf reflectance spectra could be used to rapidly estimate rice NNI, which provided a theoretical basis for monitoring rice NNI with various sensors.

    Photosynthetic and Physiological Characteristics Analysis of Yellow- Green Leaf Mutant in Wheat of Jimai5265yg
    ZHENG Wei, SHI Zheng, LONG Mei, LIAO YunCheng
    Scientia Agricultura Sinica. 2021, 54(21):  4539-4551.  doi:10.3864/j.issn.0578-1752.2021.21.005
    Abstract ( 324 )   HTML ( 47 )   PDF (907KB) ( 247 )   Save
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    【Objective】Leaf color mutants are ideal materials for studying chlorophyll synthesis, chloroplast development and photosynthesis. In order to clarify the regulation mechanism of photosynthesis and lay a foundation for the further utilization of wheat yellow-green leaf mutants, the photosynthetic physiological characteristics of wheat were studied.【Method】The wild type Jimai5265 and the mutant Jimai5265yg were used as test materials. The phenotype of leaf color was observed, the chlorophyll content and enzyme activity were measured by spectrophotometer and kit, respectively. The photosynthetic characteristics and chlorophyll fluorescence parameters were determined by the Li-6400 portable photosynthetic apparatus and PAM100 modulated chlorophyll fluorometer.【Result】The results of phenotypic observation and pigment content showed that the leaves of the mutant were yellow-green at seedling stage, and gradually changed to light green after heading stage. Leaf color of the mutant was partly recovered by shading treatment, but it was slightly lighter than the wild type, which indicated that it belonged to the mutants of light induced to promote greening. The content of chlorophyll a and b in the mutant leaves was significantly reduced, and the ratio of chlorophyll a to chlorophyll b was increased, indicating that Jimai5265yg was a typical chlorophyll deficient mutant. The light response curves and CO2 response curve displayed that surface sight-seeing quantum efficiency (AQY), light saturation point (LSP), maximum net photosynthetic rate (Pn-max), light compensation point (LCP), dark respiration rate (Rd), spindle efficiency (CE) and saturated CO2 concentration (I-sat) of the mutant was significantly higher than the wild type, indicating that the mutant had quite stable the photosynthetic mechanism and higher photosynthetic rate under the strong light; The photosynthetic gas exchange parameters and chlorophyll fluorescence kinetic parameters indicated that the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), photochemical quantum efficiency (Fv/Fm), the actual photochemical efficiency (Φ PSII) and light chemical quenching coefficient (qP) of the mutant were significantly higher compared with the wild type, which showed that Jimai5265yg had the ability of strong light energy conversion and CO2 fixation; The content of malondialdehyde (MDA) in mutant was decreased significantly, while the activity of superoxide dismutase (SOD) and catalase (CAT), as well as the content of soluble sugar and soluble protein, were significantly increased. The results indicated that the antioxidant enzyme system could reduce oxidative damage by scavenging oxygen free radicals. The damage degree of cell membrane in the mutant leaf was reduced and its stress resistance was enhanced. The activity of ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) in mutant was significantly lower, while the activity of the phosphoenolpyruvate carboxylase (PEPC) was significantly higher than that of the wild type. It was speculated that the increased activity of the C4 pathway photozyme PEPC might be the key factor for the higher net photosynthetic rate of the mutant. Post-flowering shading and exogenous spraying of Ascorbic acid and dithiothreitol DTT showed that the mutants were more sensitive to change of light intensity, and the content of AsA in leaves and the efficiency of xanthophyl cycle were higher.【Conclusion】Improvement of stomatal conductance, decrease of heat dissipation and increase of C4 pathway photozyme activity in yellow green leaf mutant Jimai5265yg were the main reasons for the increase of photosynthetic rate. These results laid the foundation for the molecular regulation of high photosynthesis properties of wheat leaf mutants.

    Effects of Continuous Application of Biochar on Chlorophyll Fluorescence Characteristics of Peanut at Different Growth Stages
    GU BoWen, YANG JinFeng, LU XiaoLing, WU YiHui, LI Na, LIU Ning, AN Ning, HAN XiaoRi
    Scientia Agricultura Sinica. 2021, 54(21):  4552-4561.  doi:10.3864/j.issn.0578-1752.2021.21.006
    Abstract ( 450 )   HTML ( 38 )   PDF (477KB) ( 368 )   Save
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    【Objective】By measuring the photosynthetic performance indexes of functional leaves of peanut at different growth stages, the effects of different amounts of biochar on electron transport, light energy absorption and redox properties of peanut leaves photosome II were studied for 9 years in a row. Meanwhile, the change trend of leaf oxygen evolution complex (OEC) damage degree was observed to provide the theoretical support for guiding peanut fertilization. 【Method】 A field experiment was conducted in 2011 with three treatments: CK (no fertilization), C15 (biochar 225 kg·hm-2), and C50 ( biochar 750 kg·hm-2). In 2019, the peanut functional leaves at different growth stages were collected, and the photosynthetic indexes of leaves after dark treatment were measured by M-PEA-2 instrument.【Result】By analyzing the chlorophyll fluorescence parameters of peanut functional leaves at different growth stages, it was found that continuous application of biochar had a significant effect on the rapid chlorophyll fluorescence kinetic curve (OJIP curve). The relative variable fluorescence intensity difference (ΔVt) of K point treated with C15 was -0.002 and -0.020 at seedling stage and flowering and needle setting stage, and -0.024 and -0.053 at pod setting stage and maturity stage. Compared with CK treatment, the relative variable fluorescence intensity of K point in functional leaves of peanut at different growth stages was significantly decreased; with the increase of biochar dosage, the decrease range of K point at flowering, needling and mature stages increased in varying degrees. The chlorophyll fluorescence parameters of peanut functional leaves had a positive response to the continuous application of biochar at the flowering, needling and maturity stages. Compared with CK treatment, the relative variable fluorescence intensity at the J-step (VJ) under C15 treatment decreased by 23.9%, the approximated initial slope of the fluorescence transient (MO) decreased by 32.1%, and the rate at which trapped excitons transfer electrons to other electron receptors downstream of QA in the electron transport chain (ΨO) increased by 25.0%.The performance index on absorption basis (PIABS ) increased by 154.6%, which alleviated the damage of oxygen releasing complex (OEC) in peanut functional leaves and improved the ability of electron transfer from primary quinone receptor (QA) to other electron receptors downstream of electron transport chain, and its effect on chlorophyll fluorescence performance increased. With the increase of biochar dosage, the effect was more obvious. At the mature stage, the effects of biochar application on chlorophyll fluorescence characteristics of peanut leaves were basically consistent with those at flowering and needling stage. The specific performance was as follows: the relative variable fluorescence intensity at the J-step (VJ) and the approximated initial slope of the fluorescence transient (MO) under C15 treatment were significantly lower than those under CK treatment 12.5% and 16.0%, respectively, and the trapped excitons transferred electrons into the electron transfer chain (QA).Compared with CK treatment, the relative variable fluorescence intensity at the J-step (VJ) decreased by 13.2%, and the approximated initial slope of the fluorescence transient (MO) decreased by 19.4%. The probability that a trapped exciton moved an electron into the electron transport chain beyond QA (ΨO) was 8.2%, and the performance index on absorption basis (PIABS) increased by 79.7%.【Conclusion】Continuous application of biochar could increase the electron transfer efficiency of photosystem II in functional leaves of peanut at the time of flowering and maturity, and improve the photosynthetic performance of functional leaves of peanut during this period.

    Research on Fine-Grained Image Recognition of Agricultural Light- Trap Pests Based on Bilinear Attention Network
    YAO Qing, YAO Bo, LÜ Jun, TANG Jian, FENG Jin, ZHU XuHua
    Scientia Agricultura Sinica. 2021, 54(21):  4562-4572.  doi:10.3864/j.issn.0578-1752.2021.21.007
    Abstract ( 427 )   HTML ( 44 )   PDF (4143KB) ( 321 )   Save
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    【Objective】Some agricultural pests trapped by the intelligent light traps show the intraspecies difference and interspecies similarity due to a variety of pest species, different pest poses and scale missing. To improve the identification rate of agricultural light-trap pests, a fine-grained image identification model of agricultural light-trap pests based on bilinear attention network (BAPest-net) was proposed to identify 19 pest species which were easily misjudged by YOLOv4 model.【Method】Firstly, according to the appearance similarity and false detection results, 19 light-trap pest species were divided into 6 similar classes. All the pest images were processed to be equal in length and width through the edge-filling operation. Then, they were scaled to a uniform size of 224×224 pixels. In order to improve the robustness and generalization ability of model, the pest images were enhanced by mirror and flipping, rotation by 180 degrees, Gaussian noise, and mean filtering. The proportions of training set, validation set, and test set in samples are 80%, 10% and 10% respectively. An agricultural light-trap pest identification model based on bilinear attention network (bilinear-attention pest net, BAPest-net) was developed to identify 19 pest species belong to 6 similar pest classes. The BAPest-net model consisted of three modules, which were a feature extraction module, an attention mechanism module and an identification module. The down-sampling step in the feature extraction module was post-handled to extract more features. The attention mechanism model could make the model focus on the local features, which could increase the identification rate and learning efficiency. The model optimizer used the stochastic gradient descent method, and the global average pooling was used in the classification module to avoid overfitting from the structure of the entire network. Finally, the five models, including VGG19, Densenet, ResNet50, bilinear model and BAPest-net, were training on the same training set and were used to test 19 light-trap pests in the 6 similar pest classes. Precision, Precision-Recall curve and average identification rate were used to evaluate the identification effects of different models on similar light-trap pests.【Result】 In five models, the BAPest-net model had the highest average identification rate of 94.9% on 19 light-trap pests in 6 similar pest classes. The bilinear model gained the second high identification rate of 90.2% and the VGG19 model had only the lowest identification rate of 82.1%. The average identification rates of Lepidoptera pests in four pest categories were greater than 95.0%. 【Conclusion】The fine-grained image identification model of agricultural light-trap pests based on bilinear attention network could automatically identify 19 agricultural light-trap pests in 6 similar pest classes and improve the automatic identification accuracy of agricultural light-trap pests.

    PLANT PROTECTION
    Effect of Bacillus subtilis NCD-2 on the Growth of Tomato and the Microbial Community Structure of Rhizosphere Soil Under Salt Stress
    SHAO MeiQi,ZHAO WeiSong,SU ZhenHe,DONG LiHong,GUO QingGang,MA Ping
    Scientia Agricultura Sinica. 2021, 54(21):  4573-4584.  doi:10.3864/j.issn.0578-1752.2021.21.008
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    【Objective】The objective of this study is to evaluate the growth promotion effect of Bacillus subtilis strain NCD-2 on tomato seedlings under salt stress, as well as the effect of strain NCD-2 on soil microbial community diversity. The results will be useful for expanding the application of strain NCD-2 in agricultural system.【Method】The pot experiments were conducted to evaluate the effects of strain NCD-2 treatment on stem length, aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight. The activity of resistance-related enzymes such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and the content of abscisic acid (ABA) were measured. High-throughput sequencing (Illumina MiSeq) technique was used to determine the bacterial and fungal community structures in the rhizosphere soil. The four treatments were set as NCD-2 strain suspension treatment (NCD0), 100 mmol·L -1 NaCl treatment (CK100), NCD-2 strain suspension + 100 mmol·L-1 NaCl treatment (NCD100) and water treatment as control (CK0).【Result】Under normal conditions, strain NCD-2 treatment significantly increased the biomass of tomato. The plant height, aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight were increased by 9.08%, 10.37%, 16.64%, 15.42% and 16.78%, respectively, when compared with the control. Under salt stress, compared to the control, the plant height, aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight were increased by 16.86%, 18.96%, 21.32%, 10.50% and 23.99% after treated with strain NCD-2, respectively. The activity of resistance-related enzymes SOD, POD, CAT and the content of ABA were increased by 50.45%, 56.18%, 29.55% and 34.60% after treated with strain NCD-2, respectively, when compared with the control. For bacteria community composition analysis, compared to CK0, the relative abundance of bacteria phylum Actinobacteria, Acidobacteria and Chloroflexi was increased by 7.28%, 15.14% and 23.03% after treated with strain NCD-2 without salt stress, respectively. The relative abundance of bacteria genus Arthrobacter, Sphingomonas, Microvirga and Streptomyces was increased by 50.88%, 15.31%, 11.32% and 16.41% after treated with strain NCD-2, respectively. Under 100 mmol·L -1 NaCl stress, compared to CK100, the relative abundance of bacteria phylum Proteobacteria, Actinobacteria, Firmicutes and Gemmatimonadetes was increased by 6.08%, 8.19%, 14.11% and 4.70% after treated with strain NCD-2, respectively. The relative abundance of bacteria genus Arthrobacter, Bacillus, Sphingomonas and Microvirga was increased by 5.54%, 31.80%, 23.39% and 23.08% after treated with strain NCD-2, respectively. For fungal community composition analysis, compared to CK0, the relative abundance of fungal phylum Mortierellomycota, Glomeromycota and Chytridiomycota was increased to 186%, 477% and 1 650% of CK0, respectively. The relative abundance of fungal genus Mortierella, Trichoderma and Preussia was increased to 186%, 108%, and 120% of CK0 after treated with strain NCD-2 without salt stress, respectively. Under 100 mmol·L -1 NaCl stress, compared to CK100, the relative abundance of fungal phylum Mortierellomycota, Glomeromycota and Chytridiomycota was increased to 345%, 154%, 921% of CK100 after treated with strain NCD-2, respectively. The relative abundance of fungal genus Mortierella was increased by 246% after treated with strain NCD-2.【Conclusion】After the treatment of strain NCD-2 under salt stress, the activity of stress-resistant enzymes and the content of ABA in tomato were increased, and the population of beneficial microorganisms in tomato rhizosphere was increased, thus improving the tolerance of tomato to salt stress and significantly increasing the growth and development of tomato.

    Transcriptome and Proteome Analysis of Bacillus subtilis NCD-2 Response to L-proline from Cotton Root Exudates
    ZHAO WeiSong,GUO QingGang,DONG LiHong,WANG PeiPei,SU ZhenHe,ZHANG XiaoYun,LU XiuYun,LI SheZeng,MA Ping
    Scientia Agricultura Sinica. 2021, 54(21):  4585-4600.  doi:10.3864/j.issn.0578-1752.2021.21.009
    Abstract ( 353 )   HTML ( 30 )   PDF (1788KB) ( 272 )   Save
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    【Objective】L-proline in root exudates of cotton is one of the key factors affecting the colonization of biocontrol microorganisms. Previous studies showed that L-proline could improve the ability of Bacillus subtilis NCD-2 biofilm formation. The objective of this study is to explore the regulatory genes related to the biofilm formation and biocontrol potential of strain NCD-2 through high-throughput sequencing technology, and to lay a foundation for further understanding the molecular interaction between cotton root exudates and biocontrol strain.【Method】Strain NCD-2 was co-cultured with L-proline at the concentration of 10 mg·mL -1 for 24 h, and it was analyzed by transcriptome (RNA-seq) and isotope labeled relative quantitative proteomics (iTRAQ). The expression of some differential genes in different metabolic pathways was verified by RT-qPCR.【Result】Transcriptome analysis showed that 1 071 differentially expressed genes (DEGs) were obtained after L-proline and NCD-2 co-culture, of which 602 genes were up-regulated and 469 genes were down-regulated. GO analysis showed that 49, 14 and 30 functional items were significantly enriched in biological process, cell component and molecular function, respectively. KEGG pathways were mainly enriched in compound metabolism, flagellum assembly, bacterial motility or chemotaxis. Proteomic analysis showed that a total of 211 differentially expressed proteins (DEPs) were detected compared to the control, of which 118 proteins were up-regulated and 93 proteins were down-regulated. GO analysis showed that 13 and 8 functional items were significantly enriched in biological process and molecular function, respectively. KEGG pathways were mainly enriched in amino acid metabolism, carbohydrate metabolism, flagellum assembly and ABC transporter. Further transcriptional-proteomics analysis revealed that 112 DEGs (or DEPs), including 38 down-regulated genes (or proteins) and 74 up-regulated genes (or proteins), were detected. GO functional items were significantly enriched in nine aspects, including nutrient reservoir activity, catalytic activity, cell membrane, localization, cellular lipid metabolic process, oxidation-reduction process, sigma factor activity, transport activity and spore formation. KEGG pathways were mainly enriched in energy metabolism, ABC transporter, antibiotic biosynthesis, flagellum assembly, motility or chemotaxis and two-component system. Twenty-six DEGs were verified by RT-qPCR. The results showed that there were some differences in the expression level, but the expression trend was basically consistent with that of RNA-seq and iTRAQ.【Conclusion】The interaction between L-proline in cotton root exudates and B. subtilis NCD-2 is a complex biological process, which depends on multiple genes in different metabolic pathway networks. It is cleared that DEGs (or DEPs) of the two-component system, antibiotic biosynthesis, energy metabolism, motility or chemotaxis, flagellum assembly and ABC transporter pathway may play an important role in the interaction between cotton root exudates and B. subtilis.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Variation of Available Phosphorus in Purple Soil and Its Effects on Crop Yield of Rice-Wheat Rotation Under Long-Term Fertilizations
    REN JiaXin,LIU Jing,CHEN XuanJing,ZHANG YueQiang,ZHANG Yong,WANG Jie,SHI XiaoJun
    Scientia Agricultura Sinica. 2021, 54(21):  4601-4610.  doi:10.3864/j.issn.0578-1752.2021.21.010
    Abstract ( 405 )   HTML ( 40 )   PDF (522KB) ( 313 )   Save
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    【Objective】Based on the analyses of soil Olsen-P variation in the purple soil and its effects on crop yield under long-term different fertilizations in the rice-wheat rotation, this paper provided a theoretical basis for efficient and rational P management in purple soil. 【Method】This study were conducted based on the 27-year rice-wheat rotation trial platform in the Purple Soil Fertility Monitoring Station of the national soil fertility monitoring network. The soil Olsen-P content and crop yields of 10 different fertilization treatments were measured and compared, including CK treatment (crops growing without fertilization), N, NP, NK, PK, NPK (treatments with different chemical nitrogen (N), phosphorus (P), potassium (K) fertilizations), and M, NPKS, NPKM, 1.5NPK+M (chemical fertilizer combined with organic manure (M) and straw return (S) treatments) from 1991 to 2018. Then, the plant P uptake per 100 kg grains yield and the recovery rate of P by different fertilizations were calculated and compared, respectively. The responses between soil Olsen-P increment and cumulative P depletion were explored. In addition, the response curve of crop yield to soil Olsen-P content in the purple soil was figured by different modelling methods. The agronomic critical value of Olsen-P content in purple soil was finally calculated. 【Result】Long-term application of P fertilizer could significantly increase soil Olsen-P content. The average annual increment of soil Olsen-P content was 0.80-2.32 mg·kg-1 in P application treatments, whereas the soil Olsen-P content of CK, N, NK and M treatments decreased year by year to a steady state. The cumulative P surpluses by the 27-year P application treatments were 244.8-698.2 kg P·hm-2, among which the cumulative P surplus of the 1.5NPK+M treatment was the highest. A significant linear correlation between cumulative soil P surplus and soil Olsen-P increment could be found in P application treatments. In detail, soil Olsen-P increased by 4.27-6.5 mg·kg-1with 100 kg·P·hm-2 cumulative surplus in P application treatments. Fertilization could significantly increase crop yields and P uptake in the long-term rice-wheat rotation system. The plant P uptake per 100 kg rice yield was 0.17-0.41 kg, whereas the plant P uptake per 100 kg wheat yield was 0.25-0.57 kg. The utilization rates of P under all treatments were 10.3%-39.7%. Four models (linear-platform model, linear-linear model, BoxLucas model, and Michelice model) were good for fitting the response of crop yield to Olsen-P content in purple soil. The agronomic critical value of Olsen-P content in purple soil of rice and wheat calculated by linear-linear model (with the highest R 2) were 13.28 mg·kg-1 and 9.93 mg·kg-1, respectively. 【Conclusion】Appropriate application of P fertilizer could significantly improve the P uptake of crop in rice-wheat rotation system on purple soil, crop yields and soil available P content. The linear-linear model was recommended to calculate the critical value of Olsen-P content in purple soil under rice-wheat rotation system. Application rates of P fertilizer should be adjusted timely according to the difference between actual soil Olsen-P content and agronomic critical value of Olsen-P content in productivity.

    Mineralization Characteristics of Soil Organic Carbon and Its Temperature Sensitivity in Wheat Field Under Film Mulching
    CAO HanBing,XIE JunYu,LIU Fei,GAO JianYong,WANG ChuHan,WANG RenJie,XIE YingHe,LI TingLiang
    Scientia Agricultura Sinica. 2021, 54(21):  4611-4622.  doi:10.3864/j.issn.0578-1752.2021.21.011
    Abstract ( 348 )   HTML ( 37 )   PDF (1152KB) ( 259 )   Save
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    【Objective】Clarify the response of dryland soil organic carbon (SOC) mineralization to long-term film mulching and its temperature sensitivity, and further understand the transformation and stabilization mechanism of SOC, providing theoretical support for dryland soil fertility and crop productivity. 【Method】Based on the dryland wheat field mulching experiment that began in 2012 in the southeastern of the Loess Plateau, soil samples of 0-20 cm layer for different mulching cultivation modes (farmer’s practice fertilization (no film mulching), monitoring fertilization (no film mulching), monitoring fertilization plus ridge mulching-furrow planting (mulching), and monitoring fertilization plus whole field film (mulching)) were collected, and then the soil samples were incubated at different temperatures (15, 25 and 35℃). The lye absorption method was used to determine the SOC mineralization rate at the 1st, 3rd, 5th, 7th, 14th, 21st, 28th, 35th and 42th days after incubation, combined with two component model to fit the cumulative mineralization and decomposition rate of soil active and recalcitrant organic carbon pool, investigate the effect of film mulching on SOC mineralization characteristics and the response of SOC mineralization to temperature.【Result】The results showed that the increasing temperature had significantly increased SOC mineralization rate, cumulative mineralization and the mineralization amount of recalcitrant organic carbon pool (Cs), but markedly decreased the temperature sensitivity (Q10) and activation energy (Ea). The mineralization rate and cumulative mineralization of SOC at 25℃ and 35℃ were about twice that at 15℃, and the mineralization of Cs increased by 93.4% and 105.3% respectively compared with that at 15℃. But Q10 (25-35℃) is 19.3% lower than Q10 (15-25℃), and Ea (25-35℃) is 68.0% lower than Ea (15-25℃). Plastic film mulching significantly increased the SOC cumulative mineralization, the mineralization of Q10, Ea and Cs. Compared with farmer fertilization treatment, both monitoring fertilization plus ridge mulching-furrow planting and monitoring fertilization plus whole field film treatments had significantly increased the SOC cumulative mineralization by 26.5%-38.6% (25℃) and 27.8%-64.4% (35℃), respectively, while monitoring fertilization plus whole field film treatment had the largest improvement. The monitoring fertilization plus whole field film treatment had also significantly increased Q10 by 28.5% (15-25℃) and 25.8% (25-35℃) and Ea by 93.4% and 193.1%, respectively. Furthermore, monitoring fertilization plus whole field film treatment had markedly increased the mineralization amount of Cs by 115.8%-2 208.2%. 【Conclusion】Therefore, film mulching accelerated the SOC mineralization in the dryland wheat field of the Loess Plateau, especially for monitoring fertilization plus whole filed filming treatment mainly increased the mineralization of recalcitrant organic carbon pool, and then significantly increased the cumulative mineralization of SOC and its temperature sensitivity.

    HORTICULTURE
    Effects of Nitrogen, Phosphorus and Potassium Dosage on the Yield, Root Morphology, Rhizosphere Microbial Quantity and Enzyme Activity of Eggplant Under Substrate Cultivation
    GAO YongBo,WANG ShiXian,WEI Min,LI Jing,GAO ZhongQiang,MENG Lun,YANG FengJuan
    Scientia Agricultura Sinica. 2021, 54(21):  4623-4634.  doi:10.3864/j.issn.0578-1752.2021.21.012
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    【Objective】The effects of different levels of NPK on eggplant yield and roots growth as well as rhizosphere microbial numbers and enzyme activities were studied, so as to provide the theoretical basis for the scientific management of NPK (nitrogen, phosphorus and potassium) for the eggplant cultivation by using substrate in solar greenhouse. 【Method】 Substrate trough with sand, slag and mushroom residue (6:3:1, V:V:V) obtained from the previous experiment and drip irrigation integrated with water and fertilizer were used. There were six treatments with fertilization dosage and 100% fertilization dosage (F4) was considered as required by the target yield (fertilization dosage of the target yield = (fertilizer required by target yield - available nutrient content in substrate ) / fertilizer utilization rate), based on the 100% fertilization dosage of F4, the fertilization dosage was reduced by 60% (F1), 40% (F2), 20% (F3) and increased by 20% (F5) and 40% (F6), respectively, and no fertilization treatment was control (CK). The effects of NPK dosage on the yield and roots growth of eggplant as well as rhizosphere microbial numbers and enzyme activity were studied. 【Result】 The eggplant yield was increased firstly and then decreased with the increasing of NPK dosage. Compared with CK, the yield per plant under all treatments increased by 101.1%-212.9%. The eggplant under F3 treatment showed the highest yield per plant and was increased by 212.9% compared with CK. Bacteria were the main microbial in the rhizosphere substrate of eggplants, followed by actinomycetes and fungi. After 90 days of transplanting, with the increasing of NPK dosage, the contents of available NPK in the substrate were increased, the numbers of bacteria, actinomycetes and fungi, the activities of sucrase, catalase and alkaline phosphatase in rhizosphere substrate were increased firstly and then decreased, the values of which were higher under F3 treatment and the urease activity was higher under F4, F5 and F6 than that under other treatments. The root activity, total root length and root surface area of eggplant were increased first and then decreased with the increasing of NPK dosage. Notably, the root activity, total root length and root surface area of eggplant under F2 treatment were increased significantly by 109.2%, 49.2% and 46.5% compared with CK, respectively. There was a significant positive correlation between bacteria number and the enzyme activities in rhizosphere substrate. The urease activity showed extremely significant positive correlation with the contents of available NPK. The activities of catalase and alkaline phosphatase showed significant positive correlation with the contents of available phosphorus and potassium. The number of bacteria, catalase activities showed significant positive correlation with root activity and yield per plant, while urease activity, available phosphorus and potassium content showed significant positive correlation with root activity and yield. The root activity showed extremely significant positive correlation with yield. 【Conclusion】 In summary, the optimal fertilizer dosage for the eggplant cultivation using the mixture of sand, slag and mushroom residue in winter-spring crop in solar greenhouse was N 180.6 kg·hm-2, P2O5 212.1 kg·hm-2and K2O 434.9 kg·hm-2, which was important to increase the yield of eggplants, root activity, rhizosphere microbial numbers and enzyme activity, and could provide a good micro ecological environment for eggplant growth.

    Comparison Analysis on Volatile Compound and Related Gene Expression in Yali Pear During Cellar and Cold Storage Condition
    YUE YingXiao,HE JinGang,ZHAO JiangLi,YAN ZiRu,CHENG YuDou,WU XiaoQi,WANG YongXia,GUAN JunFeng
    Scientia Agricultura Sinica. 2021, 54(21):  4635-4649.  doi:10.3864/j.issn.0578-1752.2021.21.013
    Abstract ( 308 )   HTML ( 31 )   PDF (1498KB) ( 217 )   Save
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    【Objective】The aim of this study was to compare the differences of fruit quality, respiration rate, ethylene production rate, electronic nose characteristics, volatile compounds and related gene expression in Yali pear between cold and cellar storage, and to further analyze the effects of two storage methods on the formation of aroma substances and its mechanism. 【Method】Postharvest Yali pear was stored in cold and cellar condition, and the fruit firmness, soluble solids content (SSC), titratable acid (TA) content, respiration rate and ethylene production rate were measured. The changes of volatile compounds during storage were measured by electronic nose, and the components and contents of volatile substances were determined by GC-MS. The expression of genes related to ethylene biosynthesis (PbACS1 and PbACO2), signal transduction (PbETR1, PbETR2, PbERS1a, PbERS1b, PbEIN3 and PbERF) and volatile compound synthesis (PbAAT1, PbADH2, PbADH3, PbADH5, PbHPL, PbLOX1 and PbLOX8) was analyzed by real-time PCR. 【Result】During cold storage of Yali pear fruit, the firmness had no obvious variation, and the SSC increased, while the TA content decreased. During cellar storage, there was a remarkable decrement in firmness and increment in TA content, but there was no obvious variation in SSC. Compared with cold storage, the respiration rate was higher and the peak of ethylene production rate appeared one month earlier under cellar storage. The results showed that the electronic nose could effectively distinguish the volatile compounds of Yali pear in different storage methods. Four sensors, including W1W, W5S, W2W and W1S, played the critical role in the identification of volatile compounds, and there were more volatile substances in cellar storage. The volatile compounds, such as aldehydes, esters, alcohols, terpenes, alkanes, etc, were found in the peel and flesh of Yali pear, and which contents were higher in peel than that in flesh. 36, 33 and 28, 24 kinds of volatile compounds were detected in peel and flesh of fruit under celler, cold storage, respectively. There were more ester compounds in cellar storage than those in cold one, among which, ethyl caproate, ethyl octanoate, ethyl butyrate, (E, Z) 2,4-decadiene ester were the main aroma substances in the peel, ethyl caproate and ethyl butyrate were the main aroma substances in flesh. The analysis on expression of genes related to ethylene and aroma compound synthesis showed that the expression of ACC oxidase (PbACO2), lipoxygenase (PbLOX1) and alcohol acyltransferase (PbAAT1) genes were significantly increased, and the expression of ethylene insensitive transcriptional regulator gene (PbEIN3) was down regulated in cellar storage in contrast to cold storage. 【Conclusion】Compared with cold storage, cellar storage condition promoted ethylene production and the expression of genes related to ethylene biosynthesis (PbACO2) and aroma compound synthesis (PbLOX1 and PbAAT1), thus, made more kinds and contents of aroma substances, and exhibited more rich aroma in Yali pear.

    FOOD SCIENCE AND ENGINEERING
    Effects of Different Drying Methods on Functional Components and Antioxidant Activity in Sweet Potato Leaves
    ZHAO Shan,ZHONG LingLi,QIN Lin,HUANG ShiQun,LI Xi,ZHENG XingGuo,LEI XinYu,LEI ShaoRong,GUO LingAn,FENG JunYan
    Scientia Agricultura Sinica. 2021, 54(21):  4650-4663.  doi:10.3864/j.issn.0578-1752.2021.21.014
    Abstract ( 322 )   HTML ( 39 )   PDF (1655KB) ( 281 )   Save
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    【Objective】 This study was aimed to explore the effects of different drying methods on functional components content and antioxidant capacity of sweet potato leaves, which could provide a theoretical basis for optimize drying process and fully utilize sweet potato leaves in the future. 【Method】 The leaves of sweet potato varieties of Tainong 71 and Shengnan were dried using five drying methods, including steam drying combined with hot air drying (SD+HAD), vacuum freeze drying (VFD), hot air drying (HAD) at 60℃, 50℃ and 40℃. The effects of different drying methods on the functional components (total phenols, total flavonoids, caffeoylquinic acids, β-carotene, vitamin D3, vitamin E, ascorbic acid, vitamin B1, and vitamin B2), antioxidant capacity (using DPPH and ABTS + radical scavenging methods) and appearance color of sweet potato leaves (chlorophyll and color value) was evaluated, and the correlation between the functional components and the correlation between functional components and antioxidant activity were analyzed. 【Result】 Free phenolic acids detected in sweet potato leaves mainly included 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, caffeic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid. The effects of five drying methods on the functional components of sweet potato leaves were different. Under VFD, the total content of free phenolic acids in Tainong 71 leaves was the highest, reaching to 38.4 mg·g-1 DW, which was 25.6 times of that under 60℃ HAD. Total phenols, total flavonoids, ascorbic acid and caffeoylquinic acids showed small differences between VFD and SD+HAD, but which were significantly higher than those of 60℃, 50℃ and 40℃ HAD. The average total phenols (64.8 mg CAE·g-1 DW) and total flavonoids (6.5 mg RE·g-1 DW) content of the two materials under VFD were the highest, which were 1.7-5.3 times and 1.7-3.8 times of that under the three types of HADs, respectively. Vitamin C was well retained under VFD (175.3-441.1 mg/100 g DW), but it was extremely low (3.4-5.7 mg/100 g DW) in the three types of HADs. Vitamin D3 and α-tocopherol had the highest content in SD+HAD. The antioxidant activity of methanol extracts from sweet potato leaves was significantly different under different drying methods (P<0.05). VFD and SD+HAD had higher free radical scavenging rates, and which were significantly higher than that under HADs. Correlation analysis showed that there was a strong correlation between total phenols, total flavonoids, total caffeoylquinic acids, vitamin D3, and α-tocopherol (P<0.01). Furthermore, a significant correlation was found between the antioxidant capacity of sweet potato leaves and the contents of total phenols, total flavonoids and each caffeoylquinic acid (P<0.01). 【Conclusion】 VFD and SD+HAD could better retain the content of polyphenols, flavonoids, vitamin D3, α-tocopherol, ascorbic acid and other functional components in sweet potato leaves, which confered strong antioxidant capacity to dried sweet potato leaves. Compared with VFD, SD+HAD had the advantages of low cost and short time-consuming, so it was the preferred drying method to retain polyphenols and flavonoids in sweet potato during practical production.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Expression Differences and Functional Analysis of Exosomes microRNA in Porcine Mature and Atretic Follicles
    CHEN HuiFang,HUANG QiLiang,HU ZhiChao,PAN XiaoTing,WU ZhiSheng,BAI YinShan
    Scientia Agricultura Sinica. 2021, 54(21):  4664-4676.  doi:10.3864/j.issn.0578-1752.2021.21.015
    Abstract ( 393 )   HTML ( 25 )   PDF (1674KB) ( 250 )   Save
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    【Objective】 To explore the regulatory role of follicular fluid Exosomes (EXs) miRNA in follicular development and atresia, the difference of miRNA expression between mature follicular fluid Exosomes (mffEXs) and atretic follicular fluid Exosomes (affEXs) were analyzed. 【Method】In this study, the follicular fluid of 4-6 mm porcine mature development and atresia follicles was extracted. Then EXs were identified by particle size analysis and Western Blot detection, respectively. the sequencing analysis of the characteristic EXs carried miRNA and functional enrichment analysis were carried out, and then the key signal pathways and differential genes were screened. Finally, mffEXs and affEXs were used as additives for granular cell culture, and Q-PCR detection technology was used to analyze the expression of key genes to verify and analyze the regulatory functions of EXs miRNA in the two types of follicular fluid in follicular development. 【Result】This study successfully separated mffEXs and affEXs. The sequencing results showed that compared with mffEXs, 90 miRNAs in affEXs were up-regulated and 220 miRNAs were down-regulated, indicating that the level of miRNA expression in follicular fluid could directly regulate follicular development. KEGG enrichment analysis showed that the differential signaling pathways of the two types of follicles were mainly concentrated in the signal pathways, such as Ras, cAMP, P53 and MAPK, which involved in the regulation of biological functions, such as oocyte development, meiosis, and granulosa cell cycle. In atretic follicles, the up-regulated expression of ssc-let-7a and ssc-miR-133a-3p potentially targeted and regulated cyclin-dependent kinase (CDK1) and insulin growth factor (IGF1), which inhibited G1 and G2/M Phase operation, and steroid hormone metabolism promoted the obstruction of granular cell cycle and the apoptosis of granular cells, causing follicular atresia; down-regulated ssc-miR-21-5p potentially targeted tumor suppressor gene (P53) and inhibited cell cycle operation to promote the apoptosis of granular cells. mffEXs and affEXs were added to granular cells cultured in vitro, and Q-PCR results showed that CDK1 was significantly up-regulated in mffEXs, while P53 was significantly down-regulated, indicating the reliability of the sequencing analysis results. These results all showed that changes in miRNA expression levels in affEXs promoted granular cell apoptosis and cell cycle arrest, causing follicular atresia. 【Conclusion】 Porcine affEXs carry miRNAs increased the regulation of CDK1, IGF1 and P53 gene expression, and inhibited the cell cycle of granulosa cells and steroid hormone metabolism and other signal pathways, causing granulosa cell apoptosis and follicular atresia.

    To Evaluate the “Two-Step” Genomic Selection Strategy in Pig by Simulation
    TANG ZhenShuang,YIN Dong,YIN LiLin,MA YunLong,XIANG Tao,ZHU MengJin,YU Mei,LIU XiaoLei,LI XinYun,QIU XiaoTian,ZHAO ShuHong
    Scientia Agricultura Sinica. 2021, 54(21):  4677-4684.  doi:10.3864/j.issn.0578-1752.2021.21.016
    Abstract ( 413 )   HTML ( 31 )   PDF (1231KB) ( 261 )   Save
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    【Background】 Since genomic selection (GS) was proposed by MEUWISSEN et al. in 2001, it has been widely used in the breeding of dairy cows, pigs, and other livestock, and has significantly improved the speed of genetic gain of various economic traits. In 2017, with the organization and coordination of the National Grazing Headquarter Station and within the framework of the National Swine Improvement Program, the genomic selection platform for pig breeding was officially launched. Although genomic selection has made positive achievements in pig breeding, and the developing of advanced genotyping technology reduced the costs dramatically, some issues were still existed, including the insufficient number of genotyped individuals in majority of core breeding farms and the inappropriate implementation processes has restricted its wide application in practice.【Objective】In combination with the actual situation of domestic pig breeding, the “two-step” strategy for genomic selection was proposed in this study, that is, the off-test evaluation and the early-stage prediction. Off-test evaluation referred to the genetic evaluation of replacement pigs by SSGBLUP after off-test, and early-stage prediction was carried out when the number of chips reached a certain scale. 【Method】 In this study, the 50 K chip datasets of three breeds consisting of Duroc, Landrace, and Yorkshire were used as the base group to simulate the large-scale population of different breeds, respectively. The four generations were simulated: the first three generations were treated as the base population, and the fourth generation as the test population, two traits with medium and low heritability was simulated for each individual. The estimated breeding values of SSGBLUP and traditional BLUP model for different traits were calculated by the pig genomic selection platform based on the HIBLUP software. The predictive performance of early-stage was evaluated according to whether the individual’s testing records have influence their genomic estimated breeding values (GEBV) in test population. 【Result】The results showed that the predictive performance of off-test evaluation and early-stage for traits with medium heritability were better than those with low heritability. The selection accuracy of SSGBLUP was better than traditional BLUP. Moreover, with the increase of the number of chips and the expansion of the population size, the prediction accuracy was higher. The early-stage predictive performance of SSGBLUP was better than that of traditional BLUP, the early-stage prediction could be carried out when the number of genotyped pigs reached about 2 000, and castrating the last 30% individuals according to GEBV could ensure that the top 1% excellent individuals would not be mistakenly eliminated. And the prediction accuracy performance was increasing with the increased number of genotyped pigs. 【Conclusion】 The “two-step” strategy pretty was conformed to the state of domestic breeding program, and was easy to implement and promote the pig breeding in China. When the number of genotyped pigs was small, off-test evaluation could be carried out to improve the accuracy of selection, as well as efficiency, to a certain extent; when the number of genotyped pigs was large, early-stage prediction could be performed by castrating the pigs on the lower rank of GEBV, which could increase the amount of testing for more excellent pigs, and could also strength the selection intensity and accelerate the genetic gain. The “two-step” strategy was in line with the actual requirements of genomic selection in pig industry. The implementation of this strategy could further promote the application of genomic selection and speed up the genetic gain in pig breeding.

    Interference in TP53INP2 Gene Inhibits the Differentiation of Bovine Myoblasts
    DU JiaWei,DU XinZe,YANG XinRan,SONG GuiBing,ZHAO Hui,ZAN LinSen,WANG HongBao
    Scientia Agricultura Sinica. 2021, 54(21):  4685-4693.  doi:10.3864/j.issn.0578-1752.2021.21.017
    Abstract ( 301 )   HTML ( 30 )   PDF (2258KB) ( 222 )   Save
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    【Background】Muscle can maintain the motor function of mammals and regulate body metabolism. Its quantity and distribution have an important influence on meat quality. The growth and development of skeletal muscle and its genetic characteristics influence and even determine the meat production and meat quality to a large extent. It is of great significance to study the growth and development of skeletal muscle. The regulatory effect of TP53INP2 on autophagy and the regulation mechanism on the differentiation of preadipocytes have been studied, but whether it affects the differentiation of bovine myoblasts has not been reported. 【Objective】 This study aims to explore the effect of TP53INP2 on the differentiation of Qinchuan bovine myoblasts in order to provide a theoretical basis for the molecular breeding of beef cattle meat traits. 【Method】 The real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the expression characteristics of TP53INP2 in different tissues of Qinchuan cattle at 24 months of age. At the same time, the expression patterns of bovine skeletal myoblasts cultured in vitro at different stages of differentiation were analyzed. TP53INP2 gene siRNA was synthesized and transfected to Qinchuan bovine myoblasts, cells were induced to differentiation 12 hours after transfection, phenotypic changes of myoblasts were observed at different time pionts, and RT-qPCR and Western Blot technologies were performed respectively to detect the expression of differentiation marker genes and proteins on the fourth day of induced differentiation. 【Result】 1. RT-qPCR results showed that the expression level of TP53INP2 was the highest in adult Qinchuan cattle Longissimus dorsi muscle tissue and the lowest in the small intestine tissue. It is higher in adult bovine heart, liver, kidney, reticulum and rumen, and lower in other tissues (compared with longissimus dorsi). 2. With the differentiation of myoblasts, the expression of this gene increased from 0 to 4 days and reached a peak on the 4th day, and then decreased. 3. After interfering with TP53INP2 siRNA in myoblasts, the number and length of myotubes in the test group were significantly lower than those in the control group. 4. RT-qPCR results showed that the expression of myoblast differentiation marker genes myogenin (MYOG) and myosin heavy chain protein 3 (MYH3) was significantly lower than that of the control group. Western Blot results showed that the protein expressions of MYOG, MYH3 and MYOD in the test group were reduced and the differences were extremely significant compared with the control group.【Conclusion】Interfering of TP53INP2 has an inhibitory effect on the differentiation of bovine myoblasts, suggesting that this gene may have an important regulatory effect on the growth and development of Qinchuan cattle muscle tissue, and it can be used for in-depth functional research for molecular breeding of beef cattle practice.

    RESEARCH NOTES
    Molecular Evolution and Function Analysis of bZIP Family in Nymphaea colorata
    YE FangTing,PAN XinFeng,MAO ZhiJun,LI ZhaoWei,FAN Kai
    Scientia Agricultura Sinica. 2021, 54(21):  4694-4708.  doi:10.3864/j.issn.0578-1752.2021.21.018
    Abstract ( 534 )   HTML ( 46 )   PDF (786KB) ( 288 )   Save
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    【Objective】The genome-wide analysis of the bZIP family in Nymphaea colorata was used to identify the bZIP (basic leucine zipper) family members in waterlily, and then which were further analyzed. This study revealed molecular evolution and function of the bZIP family in waterlily.【Method】The genome sequence of N. colorata was downloaded from Waterlily Pond database. The bZIP members in waterlily were identified by using HMMER 3.0 program, and the conserved bZIP domain was verified by using CDD program. The phylogenetic tree was constructed by the IQ-tree software. The ExPASy and SOPMA online website were performed to analyze protein structure characters. The conserved motifs were identified by using MEME program. The gene duplication events were found and visualized by the MCScan and Circos software. Transcriptome data of NcbZIP members were obtained from the NCBI website (SRA Study: SRP222853). The Pearson Correlation Coefficient (PCC) about the expression levels of the NcbZIP family members was calculated by using R software, and the network of the expression levels in the NcbZIP family was analyzed by using Cytoscape software.【Result】46 bZIP members were identified in N. colorata, and were named from NcbZIP01 to NcbZIP46 according to their chromosome distributions. The A subfamily contained the most NcbZIP members (11 NcbZIPs). There were 10 subfamilies (A, B, C, D, E, G, H, I, J and S) according to the phylogenetic analysis. The protein length in the NcbZIP family was from 101 aa to 1 898 aa, and the molecular weight ranged from 12.04 kD to 214.64 kD. The NcbZIP members from same subfamily had the similar distributions of the conserved motifs and gene structures. Waterlily had 14 chromosomes, and 46 NcbZIP members were unevenly distributed across 10 chromosomes. Chromosome 1 had the highest number of NcbZIP members. There were 10 gene duplication events in the NcbZIP family, including nine segmental duplication events and one tandem duplication event. The A subfamily had the most number of the gene duplication events (three). Based on the expression patterns in different tissues, the NcbZIP family could be divided into three groups (I, II and III). The NcbZIP members in Group I were highly expressed in all tissues, while the NcbZIP members in group II were not expressed in almost all tissues. The NcbZIP members in group III had tissue-specific expression profiles, and most of NcbZIP members in C, D and E subfamilies belonged to group III. The PPC analysis about the expression levels of NcbZIP members indicated NcbZIP45 had the highest connection with other members.【Conclusion】46 NcbZIP members were identified in Nymphaea colorata, and were unevenly distributed in 14 chromosomes. The NcbZIP family could be divided into 10 subfamilies with conserved motifs distributions and diverse expression levels. The current study could lay the foundation on the functional analysis of the bZIP family in N. colorata.