Scientia Agricultura Sinica

Physiological Changes and Integrity of ATP Synthase Subunits mRNA in Naturally Aged Cotton Seeds

SONG Ci, GU FengXu, XING ZhenZhen, ZHANG JunMing, HE WenXue, WANG TianBo, WANG YuLu, CHEN JunYing

Scientia Agricultura Sinica. 2023, 56(10): 1827-1837. doi:10.3864/j.issn.0578-1752.2023.10.001

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【Objective】Seed aging is a complex biological process, previous studies have been used to elucidate the events. However, the mechanism of seed aging is still unclear. The naturally aged cotton seeds were used as experimental materials, and the physiological and biochemical changes as well as the changes in ATP synthase mRNA integrity that occurred in cotton seed during storage were investigated in order to provide a foundation for further illuminating the aging mechanism of cotton seeds.【Method】In this study, a collection of seeds (cultivar Xinluzao 74) that had been stored for 3 and 5 years served as the experimental materials, the newly harvested seeds were used as the control (CK). The germination percentage, water absorption and viability of cotton seeds were valued by germination test between paper, low constant temperature over method, and TTC staining method, respectively; The acid value and respiratory rate of cotton seeds were determined by the acid-base titration method, and the ATP synthase activity was detected with plant ATP synthase ELISA Kit. The mRNA integrity of ATP synthase subunit α, β, γ, ε, and δ in cotton embryo was analyzed by reverse transcription blocking-double primer amplification method.【Result】Our data suggest that seed vigor dramatically decreased over storage time. After 3 and 5 years of storage, the germination percentage of cotton seeds was significantly decreased from 98.7% to 84.0% and 58.0%, respectively (P<0.05). At the initial stage of seed imbibition (the first 4 h), the water absorption rate of seeds was significantly decreased by 11.0% and 26.9%, respectively. The results of TTC staining showed that only the radicle was slightly stained in seeds preserved 5 years but not the cotyledons and other organs stained; The acid value of seeds was significantly increased by 28.4% and 40.0%, respectively (P<0.05), this indicated that severe hydrolysis of lipid occurred in seeds. Seed respiration rate and ATP synthase activity showed an increasing trend during imbibition, but the increasement was significantly decreased (P<0.05); The respiration rate of seeds was reduced by 33.3% and 49.2% after 24 hours of imbibition, and the activity of ATP synthase was decreased by 17.9% and 73.4% after 12 hours of imbibition, respectively. The results of reverse transcription blocking-double primer amplification showed that the R value of ATP synthase subunits α, β, γ, and δ mRNAs stored in seeds were significantly decreased, but the subunit ε mRNA was significantly increased. These results indicated that the integrity of the ATP synthase subunits mRNA decreased to varying degrees during the natural storage process.【Conclusion】These results showed that a prolonged storage time could reduce seed vigor; The integrity loss of ATP synthase subunit mRNAs stored in seed embryos would cause ATP synthase subunit to be impaired and ATP synthase activity declined, thus lead to a decreased production of ATP and affect seed germination capacity. This might be one of the important reasons for cotton seed aging.

Molecular Marker Assisted Identification and Application of Maize Germplasms for Maize Rough Dwarf Disease Resistance

WANG JiangHao, WANG LiWei, ZHANG DongMin, GUO Rui, ZHANG QuanGuo, LI XingHua, WEI JianFeng, SONG Wei, WANG BaoQiang, LI RongGai

Scientia Agricultura Sinica. 2023, 56(10): 1838-1847. doi:10.3864/j.issn.0578-1752.2023.10.002

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【Objective】Molecular markers tightly linked to three maize rough dwarf disease (MRDD) resistant loci were employed to identify resistant inbred lines, then the classification of heterotic groups and analysis of combining ability of these inbred lines were carried out, which proved a highly efficient way for maize MRDD resistance breeding.【Method】A recombinant inbred lines (RILs) population consisting of 263 F₉ lines was developed through single seed descent method from a segregating F₂ population by crossing a resistant inbred line K36 to a susceptible inbred line S221. The MRDD resistances of the RILs were identified in different growing environments. Meanwhile the RILs were genotyped by employing three pairs of molecular markers, 5FR, 6W53 and IDP25K which were closely linked to the three resistant loci, qMrdd2, Rmrdd6 and qMrdd8. The excellent lines with disease resistance and good agronomic traits were selected out after field evaluation. Totally 24 maize inbred lines including the elite lines were genotyped using Maize 56K SNP array, then the genetic distances between the selected lines and other elite inbred lines were calculated according to Roger's algorithm and cluster analysis was conducted to classify the heterotic groups. Meanwhile, hybrid combinations were generated and the combining abilities were tested to screen the combinations with strong disease resistance and heterosis.【Result】The inbred line K36 were homozygous resistant at the three loci, qMrdd2, Rmrdd6 and qMrdd8 while S221 were homozygous susceptible. All the 263 RILs were genotyped into 21 patterns in terms of genetic composition of the three resistant loci. The lowest DSI (0.281) appeared when all the three loci were homozygous resistant while the highest DSI (0.776) appeared when the three loci were homozygous susceptible, which were consistent with the resistant and susceptible parents (0.257, 0.623). The order of DSI from low to high value for one homozygous resistant locus was Rmrdd6 (0.396), qMrdd8 (0.478) and qMrdd2 (0.654) when the other two loci were homozygous susceptible, which showed that Rmrdd6 and qMrdd2 performed the strongest and the weakest resistance while qMrdd8 was in the middle. The variation range of genetic distance between JR2136 with the genotype of three homozygous resistant loci and other 23 inbred lines was 0.2234-0.2895, with an average value of 0.2612. The inbred line with the smallest genetic distance was C413, and the largest was Chang7-2. According to the results of cluster analysis, JR2136 was classified into Reid group, hybrid combinations with inbred lines H92 and H521 belonging to Huanggai group performed strong disease resistance and heterosis.【Conclusion】The resistance of K36 to MRDD was controlled by three loci, qMrdd2, Rmrdd6 and qMrdd8, and it had quantitative genetic characteristics and gene additive effect. Maize varieties with homozygous resistant genotypes demonstrated the strongest disease resistance. The developed molecular markers closely linked with the three resistant loci have proved valuable tools in disease-resistant breeding and screening of resistant germplasm resources. It is feasible to use molecular markers for assisted selection and gene aggregation to select highly heterotic combinations with strong disease resistance.

Effect of Phytochlorin Iron on Stress Tolerance to Waterlogging in Wheat

CHU YanMeng, MAO YingChao, CAI Jian, ZHOU Qin, DAI TingBo, WANG Xiao, JIANG Dong

Scientia Agricultura Sinica. 2023, 56(10): 1848-1858. doi:10.3864/j.issn.0578-1752.2023.10.003

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【Objective】Waterlogging stress is one of the main limiting factors for wheat production, especially in the middle and lower reaches of the Yangtze River in China. Improving the waterlogging tolerance of wheat is an important goal to achieve stable and increased yield in this region. In this study, by exploring the suitable use period and concentration of phytochlorin iron, its role in improving waterlogging stress tolerance was further evaluated mainly from the perspectives of plant photosynthesis and plant antioxidant capacity. The research results could provide the theoretical and technical support for waterlogging-resistant cultivation of wheat.【Method】Using Yangmai 16 as material, three concentrations (0.0875, 0.126, and 0.194 mmol·L^-1) of phytochlorin iron were set at anthesis and grain filling stages to screen the appropriate period and concentration for achieving a significant increase in wheat yield. Based on this, the effect of phytochlorin iron on wheat tolerance to waterlogging stress at anthesis stage was further evaluated.【Result】Compared with control, treatment with a concentration of 0.126 mmol·L^-1 phytochlorin iron at anthesis stage (A2) could significantly increase wheat grain yield by increasing the grain weight. Waterlogging stress at anthesis stage significantly reduced the chlorophyll content, net photosynthesis rate, and post-flowering dry matter accumulation and translocation to grain, resulting in grain yield reduction. However, compared with non-spraying treatment, AW2 treatment showed a higher photosynthetic pigment content, photosystem II stability, net photosynthetic rate. Meantime, the raised activities of antioxidant enzymes, reduced O₂^-production rate and H₂O₂ content, which showed correspondence with the reduced accumulation of malondialdehyde content, thus alleviated the damage of cell membrane lipid peroxidation and the yield reduction caused by waterlogging stress.【Conclusion】Spraying a concentration of 0.126 mmol·L^-1 phytochlorin iron at anthesis stage could significantly increase wheat yield. Phytochlorin iron could alleviate the plant senescence, reduce damage to PSII, enhance the activity of antioxidant enzymes, reduce the damage of cell membrane lipid peroxidation, maintain higher photosynthetic rate, reduce the degree of yield reduction, and enhance wheat tolerance to waterlogging stress.

Effects of Changing Normal and Extreme Climate States on Maize Meteorological Yield in Northeast China

ZHANG WenJing, ZHAO Jin, CUI WenQian, LI ManYao, LI E, GONG XiaoYa, YANG XiaoGuang

Scientia Agricultura Sinica. 2023, 56(10): 1859-1870. doi:10.3864/j.issn.0578-1752.2023.10.004

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【Objective】Northeast China is the main grain production base in China, which has been significantly affected by climate change in recent years. It was of great significance to understand the impact of normal and extreme climate states changes on crop yield in Northeast China for regional crop production and national food security.【Method】In this study, the maize in Northeast China was used as research object, and the main climate factors affecting maize yield were screened to analyze the effects of normal and extreme climate states changes on maize yield in 81 counties in Northeast China from 1980 to 2018.【Result】(1) The average temperature, growing degree-days (GDD), and heat degree-days (HDD) during the maize growth period showed an increasing trend, and the rising rates were 0.34 ℃·(10 a)^-1, 47.07 ℃·d·(10 a)^-1, and 5.15 ℃·d·(10 a)^-1, respectively. The precipitation showed a decreasing trend, with the rate of 7.0 mm·(10 a)^-1; the average temperature, GDD, and HDD increased from northeast to southwest, while the precipitation increased from northwest to southeast. (2) The meteorological yield of maize in Northeast China showed an increasing trend from 1980 to 1999, with a rate of 80.93 kg·hm^-2·a^-1, while it showed a decreasing trend of 46.25 kg·hm^-2·a^-1 from 2000 to 2018. In terms of spatial distribution, it showed an increasing trend from the middle to the surrounding areas. The area with high yield was concentrated in the eastern part of Heilongjiang. The change of Liaoning was the most stable, and the fluctuation range was stable in the middle area. (3) By the multiple linear regression model, HDD contributed the most to meteorological yield from 1980 to 2018, and the effect was negative, which meant extreme high temperature had the greatest impact on maize yield in Northeast China and caused maize yield reduction; GDD had a positive effect, that is, the average temperature increased maize yield, and the greater GDD, the more yield increased; the precipitation had a negative effect; the interaction between temperature and precipitation had a positive impact on maize yield in Northeast China.【Conclusion】Normal and extreme climate states changes and its impact on maize meteorological yield in Northeast China from 1980 to 2018 were as follow: the normal and extreme temperature showed an overall increasing trend, while the normal precipitation showed a decreasing trend. Extreme high temperatures and normal precipitation led to a decrease in maize yield, but the average temperature increased maize yield, and the extreme high temperature had the greatest impact. In the future, it was necessary to make full use of the average temperature state and minimize the harm caused by extreme high temperature to ensure a high-stable maize production.

Meta-Analysis of Yield Effects and Influencing Factors of Cover Crops on Main Grain Crops in China

MA JiaYu, WANG Tao, LIU XiaoLi, WANG Li, ZHANG XueCheng, WANG WenTao, KONG FanSheng, HUANG XueJun, WANG ZiYi, WANG YanDong, ZHEN WenChao

Scientia Agricultura Sinica. 2023, 56(10): 1871-1880. doi:10.3864/j.issn.0578-1752.2023.10.005

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【Objective】The objective of this study was to clarify the effect of cover cropping on the yield of main grain crops in China, and to investigate the significant influencing factors, so as to provide a scientific basis for the promotion and application of cover crops in China.【Method】A Meta-analysis including data from 903 pairwise observations from 137 publications from 1980 to 2022 was conducted to elucidate the effect of “fallow” versus “cover cropping” on yield of main grain crops. Meta regression was also conducted to explore the factors influencing the effect of cover crops on grain crops yield.【Result】Under cover crops, grain crop yields increased significantly by 12.2% compared to fallow, with wheat, rice and maize yields increasing significantly by 9.5%, 11.9%, and 19.6%, respectively. In addition, grain crop yields increased by 9.5% and 12.4% for winter and summer cover crops, respectively. Among the different types of cover crops, leguminous cover crops increased grain crop yields by 12.9% (February orchid 14.2%, Chinese milk vetch 11.8%, vetch 9.5%, pea 7.8%, soybean 7.4%), while cruciferous and gramineous cover crops increased grain crop yields by 9.3% and 8.3% (rape 7.0%, ryegrass 7.9%), respectively. However, compared with pure stands, cover crop mixtures more markedly increased grain crop yield by 17.3%. Furthermore, cover cropping years and sunshine hours significantly increased the effect of cover crops. High precipitation and temperature increased the effect of cover crops at high latitudes, while high precipitation and temperature decreased the effect of cover crops at low latitudes.【Conclusion】During the fallow period, cover crops mixtures contribute to increase grain crops yields, reduce surface exposure and make full use of solar, thermal, water and soil resources, especially during the northern summer and southern winter.

Screening of the Host Factors Interacting with CP of Citrus Yellow Vein Clearing Virus by Yeast Two-Hybrid System

BIN Yu, ZHANG Qi, WANG ChunQing, ZHAO XiaoChun, SONG Zhen, ZHOU ChangYong

Scientia Agricultura Sinica. 2023, 56(10): 1881-1892. doi:10.3864/j.issn.0578-1752.2023.10.006

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【Objective】Citrus yellow vein clearing virus (CYVCV) is one of the viruses mostly threatening the stable development of citrus industry in China, but its infection and pathogenic mechanism in citrus is still unclear. In this study, the coat protein (CP) of CYVCV was used as a bait to screen the Eureka lemon (Citrus limon Burm. f.) cDNA library, and the function of obtained host factors in the interaction between host and virus was analyzed by bioinformatics method.【Method】The total RNA of Eureka lemon leaves was extracted by the Trizol method, and then reversely transcribed to the First-Stand cDNA with SMART method, which was used as a template for obtaining ds cDNA through Long-Distance PCR. After homogenization, the ds cDNA fragments were ligated to pGADT7 plasmid vector by recombination junctions to construct the primary cDNA library of Eureka lemon. The recombinant plasmids were transfected into Escherichia coli DH10B to obtain the E. coli cDNA library of Eureka lemon, and its quality was identified. Simultaneously, the CP sequence of CYVCV was amplified by PCR and ligated into the yeast two-hybrid (Y2H) bait vector pGBKT7, and the plasmids of pGBKT7-CP and pGADT7 were co-transfected into yeast Y2H Gold. The positive yeast clones were grown on the plate of SD/-Trp, SD/-Leu-Trp, SD/-Leu-Trp/X-α-Gal and SD/-Leu-Trp-His medium, respectively, and then the growth situation of the yeast was tested to identify the toxicity of pGBKT7-CP on yeast Y2H Gold and the self-activating effect of pGBKT7-CP on the reporter gene of yeast was analyzed. Then the Y2H Gold containing bait vector pGBKT7-CP was transformed with the primary cDNA library of Eureka lemon, the co-transformed yeasts were coated on the plate of SD/-Leu-Trp, SD/-Leu-Trp-His/X-α-Gal and SD/-Leu-Trp-His-Ade/X-α-Gal medium in turn. Finally, the blue and well grown positive clones were selected. The plasmids of positive yeast clones were extracted and sequenced. The candidate genes were preliminarily compared in the GenBank, and the interacted protein factors were annotated and the protein’s biological functions were analyzed with gene ontology (GO) pathway of Uniprot online websites. According to the results of the analysis, candidate factors that may be involved in host disease resistance or symptom development were selected, their CDS full-length sequences were amplified and constructed into the target vector pGADT7 and then were verified with pGBKT7-CP by one for one in yeast, respectively.【Result】The titer of the Eureka lemon-E. coli cDNA library was 1.02×10⁸ cfu/mL, and it demonstrated that the cDNA library measured up to the experiment standard. The bait vector of pGBKT7-CP was constructed, which had no ability to activate the reporter gene and had no virulence to the yeast. The 41 positive clones were finally selected by using the SD/-Leu-Trp-His-Ade/X-α-Gal medium. After sequence similarity comparison, removing the repetitive sequences, the vector sequences and the frameshift sequences, the 32 host factors that interacted with CYVCV CP were screened. The GO pathway annotation results indicated that these host factors were involved in several chloroplast-related biological processes, including photosynthesis, metabolic process, carbohydrate metabolic process, response to light stimulus, etc. The molecular functions of the 32 host factors were multiple, including catalytic activity, hydrolase activity, transferase activity, protein binding, DNA-binding transcription factor activity, and translation factor activity, etc. Moreover, the cell components of the 32 host factors were involved in the cytoplasm, membrane, chloroplast, thylakoid, nucleolus, and golgi apparatus, etc. Validation of one-to-one yeast two-hybrid crosses of 14 important proteins selected from candidate host factors with CP showed that CP interacted with all 14 proteins.【Conclusion】The cDNA library of Eureka lemon was constructed successfully, and 32 host factors of Eureka lemon interacted with CYVCV CP were preliminarily screened. According to the results, the function of the important proteins was analyzed. It was presumed that CYVCV CP affects photosystem stability, thylakoid structure and chlorophyll synthesis by interacting with multiple chloroplast-related proteins such as oxygen-evolving enhancer protein 2 (PsbP), chlorophyll a-b binding protein 8 (Lhca3) and ribulose bisphosphate carboxylase small subunit (RbcS), resulting in reduced photosynthesis and damaged chloroplast morphology and function. The interaction of CP with these chloroplast-associated factors was separately verified one-to-one in yeast, which will provide a theoretical basis for revealing the molecular mechanism of CYVCV CP in viral pathogenicity.

Viruses Identification and Their Gene Sequences Analysis Infecting Aucuba japonica var. variegata

LIANG HaiWen, LAN PingXiu, LIU QinHai, TAN GuanLin, CHEN XiaoJiao, ZHAO Yan, LI Fan

Scientia Agricultura Sinica. 2023, 56(10): 1893-1904. doi:10.3864/j.issn.0578-1752.2023.10.007

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【Objective】The objective of this study is to identify the viruses infecting Aucuba japonica var. variegata plants with symptoms of yellowing and shrinking which collected from Yunnan and Hainan provinces.【Method】Firstly, RNA sequencing was used to detect the viruses infecting A. japonica var. variegata plants from Kunming, Yunnan Province. Then, based on the RNA sequencing results, RT-PCR was used to detect these viruses in 73 A. japonica var. variegata field samples collected in Kunming of Yunnan Province and Haikou of Hainan Province, and the related virus gene fragments were amplified, sequenced and analyzed.【Result】RNA sequencing results showed that 5 viruses were detected from the pooled diseased plants, such as broad bean wilt virus 2 (BBWV2), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pea enation mosaic virus 1 (PEMV-1), and pea enation mosaic virus 2 (PEMV-2). However, only BBWV2, BYMV, CMV and PEMV-2 were detected in the 73 A. japonica var. variegata samples from Yunnan and Hainan. Among them, BBWV2 was detected in the A. japonica var. variegata samples both from Yunnan and Hainan, with total detection rate of 11.0%, while BYMV, CMV and PEMV-2 were only detected in the Yunnan samples with detection rate of 5.5%, 1.4% and 1.4%, respectively. However, neither PEMV-1 nor the co-infections among BBWV2, BYMV, CMV and PEMV-2 was detected in these 73 A. japonica var. variegata samples from Yunnan or Hainan. In order to further analyze the molecular variation of BBWV2, BYMV, CMV and PEMV-2 aucuba isolates with the related virus isolates from other host plants, the 490 nt of small cp of BBWV2 (accession number OQ137565, OQ137566), 499 nt of the core cp region of BYMV (accession number OQ137568), 880 nt of the cp of CMV (accession number OQ137567), and 800 nt of the core RdRp of PEMV-2 (accession number OQ137569) were amplified from the A. japonica var. variegata samples infected with the above 4 viruses, and then the amplified gene sequences were analyzed, respectively. Sequence comparison analysis results showed that BBWV2 Yunnan aucuba isolate shared 85.9% nt identity with Hainan aucuba isolate, the BBWV2 Yunnan and Hainan aucuba isolates shared 79.8%-94.5% and 80.2%-92.0% nt identity with other BBWV2 isolates retrieved from GenBank, respectively. BYMV, CMV, and PEMV-2 aucuba isolates shared 79.8%-99.0%, 71.5%-99.4% and 84.9%-98.0% nt identity with other corresponding viruses in GenBank, respectively. Phylogenetic trees based on the gene nucleotide sequences of the above viruses revealed that the BBWV2 Yunnan and Hainan aucuba isolates were grouped into subgroup a and subgroup b in BBWV2 group I, respectively, and the BYMV Yunnan aucuba isolate showed a closest relationship with Iranian (accession number MN241051, MN241060) and Iraqi (accession number JQ026005) broad bean isolates. Moreover, the CMV Yunnan aucuba isolate was clustered into CMV IB subgroup of group I, but showed a distant relationship with other CMV isolates in the same subgroup. While PEMV-2 Yunnan aucuba isolate showed a closest relationship with Yunnan pea isolates and clustered into the same group, and had a distant relationship with other PEMV-2 isolates.【Conclusion】To our knowledge, this is the first record of virus infecting A. japonica var. variegata, and also the first report of BBWV2, BYMV, CMV and PEMV-2 infecting A. japonica var. variegata as well as the plants in genus Aucuba. Significant molecular variation was detected among the BBWV2, BYMV, CMV and PEMV-2 aucuba isolates with the related virus isolates from other plants worldwide.

Hyperspectral Prediction of Organic Matter in Soils of Different Salinity Levels in the Yellow River Delta

HOU HuaGang, WANG DanYang, MA SiQi, PAN JianJun, LI ZhaoFu

Scientia Agricultura Sinica. 2023, 56(10): 1905-1919. doi:10.3864/j.issn.0578-1752.2023.10.008

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【Objective】The aim of this study was to investigate the spectral response of soil organic matter and salt, to analyze the effects of different salt content on soil organic matter prediction models, and to establish a rapid and effective hyperspectral prediction model for organic matter content in saline soils.【Method】In this study, according to different salinity contents for indoor hyperspectral measurements, the soil samples were divided into four groups of non-saline (SA), slightly saline (SB), moderately saline (SC), and heavy saline (SD). Then, ANOVA was used to explore the degree of organic matter and salinity spectral response of soils with different salinity degrees respectively. The raw spectra reflectances (raw spectral reflectance, R) were subjected to first order differential reflectance (first order differential reflectance, FD), continuous statistical removal (continuous statistical removal, CR), logarithmic (logarithmic, Log) and multiple scatter correction (multipication scatter correction, MSC) transformations were applied to the raw spectra reflectance; finally, three soil organic matter prediction models, namely multiple linear regression (multiple linear regression, MLR), partial least squares regression (partial least squares regression, PLSR) and support vector regression (support vector machine, SVR), were constructed based on four sets of samples of saline soils combined with the four transformed spectra, respectively.【Result】Soil organic matter and salinity had significant spectral response in the range of 400-900 nm and the change pattern were the same basically, and the sensitive bands of the two overlap. Modeling by dividing different salinity levels could improve the prediction accuracy of soil organic matter, but the prediction accuracy of the model decreased with the increase of salinity content. FD treatment could better highlight the difference of spectral characteristics and improved the correlation between organic matter content and spectral reflectance. Comparing the results of the three models, the highest accuracy of the soil organic matter prediction model was established using FD treatment combined with SVR, and the coefficients of determination R² of the optimal result modeling set and validation set were 0.86 and 0.82, respectively, the root mean square error RMSE was 2.71 and 2.96 g·kg^-1, respectively, and the ratio of prediction to deviation RPD was 2.42.【Conclusion】Soil salinity and organic matter overlapped in the sensitive bands near the visible wavelength (400-900 nm), and the accuracy of the organic matter prediction model could be effectively improved by classifying different salinity levels.

Variation Characteristics of Soybean Yield and Soil Nitrogen Distribution in Brown Soil Under Long-Term Fertilization

LIU YuYing, SHEN Feng, YANG JinFeng, CAI FangFang, FU ShiFeng, LUO PeiYu, LI Na, DAI Jian, HAN XiaoRi

Scientia Agricultura Sinica. 2023, 56(10): 1920-1934. doi:10.3864/j.issn.0578-1752.2023.10.009

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【Objective】The aim of this study was to explore the evolution, stability and sustainability of soybean yield and characteristics of soil nitrogen (N) distribution in brown soil under different fertilization in a long-term experiment, so as to provide a scientific basis for making reasonable fertilization managements and realizing sustainable and green production of soybean in northeast region with brown soil.【Method】This study was based on the long-term fertilization experiment with brown soil, which began in 1979 with the crop rotation system of maize-maize-soybean, and 12 of the treatments were selected, including 4 chemical fertilization treatments (no fertilization (CK), single N fertilizer (N), N and phosphorus (P) fertilizer mixed application (NP), and N, P, and potassium (K) fertilizer compound application (NPK) ), single application of manure at a low rate (M₁), manure at a low rate combined with chemical fertilizer (M₁N, M₁NP, M₁NPK), single application of manure at a high rate (M₂), and manure at a high rate combined with chemical fertilizer (M₂N, M₂NP, M₂NPK). The evolution of soybean yield under long-term different fertilization and the effects of 39-year crop rotation fertilization on nitrogen uptake of soybeans and soil nitrogen accumulation distribution at harvest stage were analyzed.【Result】Compared with the CK treatment, the average yield of soybean under each fertilization treatment was significantly improved, and those under the treatments with manure at low and high rate were higher than those at treatments with chemical fertilizer alone, and the average yield under M₁NPK and M₂NPK treatments were the highest with 3 147 and 3 238 kg·hm^-2, respectively, which were 9.5% and 12.7% higher than that at NPK treatment. The results of the grey-linear regression showed that application with manure simply or combined with chemical fertilizer significantly increased the interannual yield compared with that with chemical fertilizer alone. The variation coefficient of soybean yield at treatments with manure at the low rate was the lowest with a high yield stability. The yield sustainability index (YSI) was higher ranging from 0.41 to 0.51, which was higher than that under treatments with manure at the high rate. Combined application of manure increased the contribution of fertilization to soybean yield, but without significant difference between treatments with manure at the low and high rate. After application of manure for 39 years, the soybean N uptake increased compared with the treatments with single chemical fertilizer, which was the highest at M₁NPK treatment being 314.2 kg·hm^-2. With application of manure at the low rate, soil mineral N mainly accumulated in 0-60 cm soil layers, and its accumulation at 60-100 cm soil depths was low. The mineral N accumulation in the 0-80 cm soil layers with application of manure and chemical fertilizer were higher than those under M₁ treatment, which would provide available N for crop, but the mineral N in the 80-100 cm soil layer was lower than that in the upper soil, which reduced the risk of N leaching. Among them, the mineral N accumulation in the 0-60 cm soil layers was the highest at M₁NPK treatment, and the 60-100 cm soil layer showed a continuous decrease trend with the increase of soil depth, while the M₂NPK treatment of the block with manure at the high rate showed a trend of first decreasing and then increasing. The soil total N and microbial biomass N were increased in the top 20 cm soil layer after fertilization with manure and chemical fertilizer for 39 years, which were higher than that in the 20-40 cm soil depth. Compared with NPK treatment, the total N concentration in the 0-20 cm soil layer under M₁NPK and M₂NPK treatments increased by 13.9% and 5.5%, respectively, where the microbial biomass N concentration increased by 32.6% and 92.1%, respectively.【Conclusion】Long-term fertilization affected crop yield, N uptake, and soil N distribution. In the maize-maize-soybean rotation system in the brown soil area of Northeast China, the application of N, P, and K fertilizer combined with manure at a low rate (13.5 t·hm^-2) in the maize season, and the lonely application of N, P, and K fertilizer in the soybean season changed the soil N distribution and accumulation, and thus influenced the soybean N uptake, increased the soybean yield, improved the yield stability and sustainability. The increase of soil total N and microbial biomass N concentration at soybean harvest under long-term application of manure at a low rate increased the soil N supply, meanwhile the reduction of mineral N accumulation in deep soil reduced the risk of N loss by leaching, which was conducive to the sustainable and green production of soybean and was a reasonable fertilization method for this rotation system.

Effects of Biochar Combined with Dicyandiamide on Greenhouse Gases Emissions from Facility Vegetable Soil

SONG BoYing, GUO YanJie, WANG WenZan, LÜ ZeNan, ZHAO YuQing, LIU Lu, ZHANG LiJuan

Scientia Agricultura Sinica. 2023, 56(10): 1935-1948. doi:10.3864/j.issn.0578-1752.2023.10.010

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【Objective】This paper aimed to explore the comprehensive effects of biochar, dicyandiamide (DCD) and their combined application on the greenhouse gas (N₂O, CO₂ and CH₄) emissions from facility soil, so as to provide a scientific basis for reducing the greenhouse gas emissions and green development of facility vegetable production system.【Method】In this study, the facility vegetable production system was used as the research object, and a total of six treatments were set up, including no nitrogen application (CK), traditional nitrogen application (CN), recommended nitrogen application (RN), recommended nitrogen application+biochar(RNB), recommended nitrogen application + DCD (RND), and recommended nitrogen application+biochar+ DCD(RNBD). A pot experiment method was applied to analyze the effects of soil greenhouse gas emissions, and the difference in greenhouse gas intensity (GHGI) and global warming potential (GWP) under different treatments.【Result】Compared with the CN treatment, the rape yield decreased by 2.9%-29.3% under the recommend nitrogen treatments (RN, RN, RND and RNBD). However, under the same nitrogen application rate, the rape yield increased by 34.4% in the treatment of recommend nitrogen combined with biochar and DCD (RNBD), indicating that biochar and DCD showed a synergistic effect on rape yield increase (P<0.05). The recommend nitrogen treatments reduced the soil N₂O emissions by 29.4%-76.5% in comparation with the CN treatment, especially the RND treatment showed the best effect. However, the recommend nitrogen treatments showed little effect on soil CO₂and CH₄ emissions. Compared with the CN treatment, the total GWP under the recommended nitrogen treatments decreased by 4.3%-51.2%, and the RND treatment showed the best emission-reduction effect. In terms of GHGI, the difference among the recommended nitrogen treatments was not significant (P>0.05), and the RND treatment also showed the best emission-reduction effect.【Conclusion】Under the same nitrogen application rate, the application of biochar alone or DCD alone had little effect on rape yield, but the combination of biochar and DCD could significantly increase the rape yield. Additionally, the combination of biochar and DCD could reduce the cumulative greenhouse gas emissions and GWP, but it was not superior to single application of DCD in the facility vegetable field.

Comprehensive Fruit Quality Evaluation and Suitable Areas Selection of Newhall Navel Orange in China

CHEN ZhiMin, CHEN XiaoLin, TAN ZhenHua, CHEN ZhaoXing, SHEN DanDan, MA YanYan, ZHENG YongQiang, YI ShiLai, LÜ Qiang, XIE RangJin

Scientia Agricultura Sinica. 2023, 56(10): 1949-1965. doi:10.3864/j.issn.0578-1752.2023.10.011

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【Objective】This study aimed to establish a comprehensive evaluation model in fruit quality, clarify the comprehensive grade of fruit quality and their corresponding meteorological characteristics from different Newhall Navel orange (Citrus sinensis [L.] Osb. cv. Newhall) orchards in China, so as to provide a reference for the ecological environment adaptability and suitable planting.【Method】Twenty three Newhall orchards in different ecological environments of China were selected to measure fruit external and internal quality indicators. Correlation analysis, principal component analysis and cluster analysis were used to identify the core indicators, and the comprehensive evaluation models of fruit quality were established by analytic hierarchy process (AHP), principal component analysis (PCA) and fuzzy comprehensive appraisal (FCA), respectively. The optimal algorithm model and fruit grade classification threshold were determined and verified with sensory quality evaluation. Meanwhile, the comprehensive quality grades and the characteristics of corresponding ecological factors of Newhall navel orange fruit from different producing areas were explored.【Result】The fruit comprehensive quality of 23 Newhall orchards showed obvious regional characteristics. The fruit in southern Jiangxi and southern Hunan had higher total soluble solid and solid acid ratio, while the fruit in western Hunan and the upper and middle reaches of the Yangtze River had better fruit external color indexes and higher titratable acid. The quality indexes were correlated to varying degrees. Five core indexes were selected by principal component analysis combined with cluster analysis, including comprehensive color index, fruit weight, soluble solids, solid acid ratio, and vitamin C content. At the same time, AHP model was determined as the comprehensive evaluation model of fruit quality with the best fitting degree of sensory quality index: Y (comprehensive value) = 0.06× comprehensive color index + 0.26× single fruit weight +0.16× soluble solid content +0.42× solid acid ratio +0.11× vitamin C content (standardized value). The comprehensive fruit quality indicators of different Newhall orchards were ranked, and the classification threshold was determined as follows: ≥0.60 was the first-class orchards, mainly concentrated in southern Jiangxi, southern Hunan and eastern Guangdong, with active accumulated temperature, effective accumulated temperature, maximum sunshine hours and surface temperature; 0.45-0.60 was the second-class orchards, mainly concentrated in northern Guangxi and western Fujian, characterized by higher temperature accumulation and rainfall; 0.30-0.45 was the third-class orchards, mainly concentrated in western Hunan and the middle and upper reaches of the Yangtze River, with relatively lower rainfall and temperature accumulation; <0.30 was the fourth-class orchards, mainly distributed in southern Zhejiang, with the highest rainfall.【Conclusion】The core indicators of the comprehensive quality of citrus fruits were identified by PCA, and the AHP model with the optimal for the Newhall fruit comprehensive evaluation by combining the eigenvalues of each principal component to achieve automatic assignment of AHP judgment matrix. The ecological factors of different grades of orchards were significantly different. These results provided algorithms and data support for the development of the decision system based on "suitable planting" of citrus varieties in different ecological environment.

Identification of the VvmiR164s-VvNAC100 Action Module and Analysis of Their Expressions Responsive to Exogenous GA During Grape Ovary Development

WANG Fei, XIAO YingKe, XUAN XuXian, ZHANG XiaoWen, LIU Fei, ZHA ZiXian, DAI MengTong, WANG XiCheng, WU WeiMin, FANG JingGui, WANG Chen

Scientia Agricultura Sinica. 2023, 56(10): 1966-1981. doi:10.3864/j.issn.0578-1752.2023.10.012

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【Objective】The aim of this study was to identify grapevine miR164s (VvmiR164a/b/c/d) and their target genes, and to elucidate the regulatory roles of VvmiR164s and their target genes during exogenous GA-induced grape parthenocarpic process.【Method】Using ‘Wink’ grapes (Vitis vinifera L. Wink) as the test material, miR-RACE, PCR, RLM-RACE and PPM-RACE, qRT-PCR and bioinformatics were used to analyze the spatio-temporal expression of VvmiR164s-VvNAC100 module in response to exogenous GA and its potential functions during grape parthenocarpic process.【Result】Gibberellin application on ‘Wink’ grapes before flowering induced parthenocarpy, leading to seedless berries. The precise sequence of VvmiR164a/b/c/d was cloned and identified, and four of its target genes were predicted, including VvNAC100-1, VvNAC100-2, VvNAC098, and VvNAC021. Combining the degree of match with the comprehensive analysis of the previous data, this work focused on the analysis of the target gene VvNAC100-2, which was named as VvNAC100. The VvNAC100 cleavage site was located at position 9 and position 11 of the 5′ end of miRNA, with a cleavage frequency of 17/20 and 11/20, respectively, and was localized on Chr14, encoding 363 amino acids and containing a NAM. The VvNAC100 protein is highly conserved in amino acid sequence and functionally similar to other species, among which it is more closely related to species such as pepper and tobacco. The promoters of VvMIR164a/b/c/d and its target gene VvNAC100 both contain multiple hormone-acting elements, suggesting that it might be involved in the regulation of grape growth and development by responding to mult-hormones. The RT-qPCR results showed that the expression level of VvmiR164b tended to decrease as the grape ovary developed, while its target gene VvNAC100 showed an increasing expression trend in the early stage of ovary development with a certain negative correlation. On the other hand, VvmiR164a/c/d showed a similar expression pattern with VvNAC100 with a certain positive correlation. However, after GA treatment, the expression of VvmiR164a/c/d was highly significantly increased during parthenocarpy in the grape ovary, which also significantly suppressed the expression of VvNAC100 during this period, promoting a negative correlation between VvmiR164a/c/d-VvNAC100 their expression levels. VvmiR164b showed a decreasing trend after GA treatment, and it’s the expression levels showed some positive correlation with those of VvNAC100, indicating that GA treatment promoted the negative regulation of VvmiR164a/c/d on VvNAC100, but repressed the negative regulation of VvmiR164b on VvNAC100.【Conclusion】VvNAC100 was the true target gene of VvmiR164 family. During grape parthenocarpy, GA induced the negative regulation of VvmiR164a/c/d on the target gene VvNAC100 and suppressed that of VvmiR164b on the target gene VvNAC100. VvmiR164a/c/d were the main effectors of the VvmiR164 family involved in modulation of GA-induced grape parthenocarpy.

Fractionation Effect of Stable Carbon and Nitrogen Isotope Ratios in Yak Dairy Products Processing

LI JiRong, LIU Xin, WANG Jun, CAO XiaoGang, CI Dun

Scientia Agricultura Sinica. 2023, 56(10): 1982-1993. doi:10.3864/j.issn.0578-1752.2023.10.013

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【Background】Stable isotope fingerprinting technology has been widely adopted in the origin traceability study of dairy products. However, most of them are focused on comparing the differences between the stable isotopes of raw milk and milk products. Nevertheless, the fractionation effect of stable isotopes on dairy products processing and the application of stable carbon and nitrogen isotopes for origin tracing of yak dairy products are still unclear.【Objective】In this study, yak yogurt and yak milk dregs were used as the study subjects to determine the changes in stable carbon and nitrogen isotope and the fractionation coefficients and correlations of yak dairy products at key points during processing, to investigate the stable carbon and nitrogen isotope characteristics of yak dairy products from different origins, so as to provide the theoretical and technical supports for origin traceability of yak dairy products.【Method】A total of 196 samples were collected from the Nerong and Jiali counties of Nagqu City, Tibet Autonomous Region, obtain five key sampling points for yogurt processing (yak milk, yak milk boiled for 5 min, sample after strain addition, fermentation at 40 ℃ for 6 h, and yogurt) and four key sampling points for milk dregs processing (yak milk, skimmed yak milk, skimmed yak milk boiled for 10 h, and milk dregs). The stable carbon and nitrogen isotope ratios were determined using an elemental analysis isotope ratio mass spectrometer (EA-IRMS). The differences and correlations between the stable carbon and nitrogen isotopes at the key sampling points for yogurt and milk dregs processing were determined using one-way ANOVA comparative analysis and Pearson correlation analysis, respectively. Furthermore, the differences in stable carbon and nitrogen isotopes between yogurt and yak milk and milk dregs and yak milk with different origins were determined using a two-factor ANOVA.【Result】The fractionation of stable carbon and nitrogen isotope during yogurt processing was as follows: δ¹³C_{yak milk}>δ¹³C_{40 ℃ fermentation for 6 h, yak yogurt}>δ¹³C_{samples after adding strain}, fractionation coefficient between 0.9996 and 1.0009, Δ_{Yak milk-yak yogurt} was 0.48‰; δ¹⁵N_{boiling 5 min yak milk, 40 ℃ fermentation for 6 h, yak yogurt}>δ¹⁵N_{yak milk}, fractionation coefficient was between 0.9993 and 1, and Δ_{Yak milk-yak yogurt} was -0.61‰. The correlations between the stable carbon and nitrogen isotopes at some key sampling points were significant. During milk dregs processing, δ¹³C_{yak milk, boiled 10 h skimmed sample, yak milk dregs}>δ¹³C_{Skimmed yak milk}, fractionation coefficient was between 0.9995 and 1.0005, Δ_{Yak milk-yak dregs} was 0. A significantly negative correlation was observed in δ¹³C at some key sampling points, while no significant difference was observed in δ¹⁵N for each key point sample and the fractionation values were 0. The stable carbon and nitrogen isotopes of dairy products from different origins significantly differed, with δ¹³C and δ¹⁵N being enriched in yak dairy products from Nerong County compared to Jiali County.【Conclusion】The fractionation of δ¹³C and δ¹⁵N was observed during yak dairy products processing. The addition of strains, fermentation, and centrifugal defatting processes resulted in different δ¹³C ratios, while heating induced changes in the sample δ¹³C and δ¹⁵N. Although stable isotope fractionation occurred during yak dairy products processing, its influence was less than the origin. Therefore, the stable carbon and nitrogen isotopes could be applied to trace the origin of yak dairy products.

Identifying Quantitative Trait Loci Associated with Teat Number of Pig by Genomic Analysis

YIN YanZhen, HOU LiMing, LIU Hang, TAO Wei, SHI ChuanZong, LIU KaiYue, ZHANG Ping, NIU PeiPei, LI Qiang, LI PingHua, HUANG RuiHua

Scientia Agricultura Sinica. 2023, 56(10): 1994-2006. doi:10.3864/j.issn.0578-1752.2023.10.014

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【Objective】The purposes of this study were to analyze the variation of teat number, to explore the quantitative trait locus (QTL) and candidate genes related to teat number, and to provide important molecular markers for the breeding of pig teat number.【Method】This study accurately measured left, right, total teat number of 709 Suhuai pigs (335 fattening pigs and 374 breeding pigs). Fattening pigs were selected for 80K chip genotyping and the heritability and genomic estimated breeding value (GEBV) of left, right and total teat number were calculated by chip data. Based on the rank of GEBV and phenotype of teat number, the top 10% individuals and the bottom 10% individuals were selected for Fixation Index (F_ST) analysis to detect highly differentiated loci. Then, the loci associated with teat number were identified by genome wide association analysis (GWAS) and loci which were highly differentiated and significantly associated with teat number were selected as candidate loci. Genes located near candidate loci and related to teat number after functional annotation were selected as candidate genes. Finally, the association analyses between the most significant candidate loci on each chromosome and teat number of 709 Suhuai pigs were performed to verify the significance of the above loci.【Result】The variation coefficients of left, right and total teat number of Suhuai fattening pigs were 10.20%, 9.26% and 8.50%, respectively, and the heritability were 0.212, 0.257 and 0.312, respectively. Based on F_ST and GWAS analyses, a total of 20 candidate loci on Sus scorfa chromosomes (SSC) 7, 13, 16 and 18 for teat number were identified and these candidate loci could explain 5.49%-8.03% of the phenotypic variance. Among them, locus rs80894106 on SSC7 associated with total teat number was consistent with the reported candidate locus of total teat number based on Large white and Duroc pig populations, but candidate loci rs81444134 (26.51 Mb, SSC13) and rs81233299 (8.13 Mb, SSC18) of left teat number were newly discovered loci related to teat number. Interestingly, candidate loci of left, right and total teat number were mainly concentrated in the 6.36-10.66 Mb interval on SSC16; Linkage disequilibrium (LD) analysis found that candidate loci in 7.47-8.27 Mb interval fit into a 795 kb haplotype block, and this haplotype block was a newly discovered candidate area that affected teat number; rs337606862 (7.47 Mb) in the haplotype block was the most significantly SNP associated with the left and total teat number, and three loci in the haplotype block were all located on the intron of cadherin 18 (CDH18) gene; CDH18 gene encoded type II cadherin, and cadherin was related to the identification, sorting, proliferation, apoptosis of cells in developing tissue and the occurrence of breast cancer. Thus, CDH18 might be a new candidate gene that affected pig teat number. In addition, the most significant loci rs81444134, rs80894106, rs337606862 and rs81233299 on 4 chromosomes were genotyped in 709 Suhuai pigs in this study. After association analysis, these loci were significantly associated with teat number, and could be used as potential molecular markers for the selection of teat number.【Conclusion】In this study, 20 loci significantly related to teat number were identified in Suhuai pig population by genome analysis. Among them, 26.51 Mb on SSC13 and 8.13 Mb on SSC18 were new candidate QTLs for teat number. The 7.47-8.27 Mb on SSC16 was also a newly discovered candidate QTL for teat number, and CDH18 gene in this interval might be a new candidate gene that affected the formation of pig teat.

Role of FATP1 in Promoting Lipid Deposition in Goat Intramuscular Adipocytes

LI Qi, YANG ChangHeng, WANG Yong, LIN YaQiu, XIANG Hua, ZHU JiangJiang

Scientia Agricultura Sinica. 2023, 56(10): 2007-2020. doi:10.3864/j.issn.0578-1752.2023.10.015

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【Background】Fatty acid transporter 1 (FATP1) can promote the uptake of fatty acids in mammals. This process is very important to maintain the balance of lipid metabolism, and also has an important impact on the meat quality of livestock.【Objective】The aim of this study was to obtain the CDS sequence of goat FATP1 gene, to detect the expression of FATP1 gene in different tissues of goats, and to explore its effect on lipid metabolism of goat intramuscular adipocytes, so as to provide a reference for further revealing the mechanism of FATP1 gene in goat lipid metabolism, which can provide a theoretical basis for genetic and breeding improvement of goats.【Method】The CDS of goat FATP1 gene was cloned by real-time fluorescence quantitative PCR (RT-PCR), its biological characteristics, such as hydrophobicity, transmembrane region and signal peptide, were analyzed by online tools, and its amino acid sequence phylogenetic tree was constructed. The expression level of FATP1 gene in different goat tissues was detected by RT-qPCR and its tissue expression pattern was constructed. The constructed eukaryotic expression vector and screened siRNA were used to overexpress and interfere with FATP1 in goat intramuscular adipocytes, the effects of FATP1 gene overexpression and interference on lipid deposition in goat intramuscular adipocytes were detected by oil red O staining and triglyceride determination, and the effects of FATP1 gene overexpression and interference on the expression of genes related to lipid metabolism were further explored by RT-qPCR.【Result】The CDS of FATP1 gene was 1 941 bp, encoding 646 amino acids residues. It was predicted that its molecular formula was C₃₁₉₆H₅₀₂₆N₈₈₄O₈₉₈S₂₅, and the protein was a basic hydrophobic stable protein. Phylogenetic tree analysis of amino acid sequence showed that goat FATP1 was closely related to sheep. RT-qPCR showed that the expression of FATP1 gene was the highest in goat small intestine. Oil red O staining and triglyceride determination showed that the number of lipid droplets and triglyceride content in goat intramuscular adipocytes increased after overexpression of FATP1, but the opposite results were obtained after interference with FATP1. After overexpression of FATP1 in goat adipocytes, the expression levels of fatty acid synthesis, transport and other related genes AGPAT6(P<0.01), PLIN1(P<0.01), DGAT2(P<0.01), FADS2(P<0.01), FADS1(P<0.01), ACSL1(P<0.01) and ELOVL3 (P<0.05) increased significantly, while the expression level of lipolysis related genes ACOX1 (P<0.01) decreased significantly. After interfering FATP1, the expression of fatty acid transport, elongation and other related genes SCD5 (P<0.01), FABP3 (P<0.01) and ELOVL3 (P<0.05) decreased significantly, and the expression of lipolysis related genes ACOX1 (P<0.01) and CPT1B (P<0.05) increased significantly.【Conclusion】FATP1 might significantly promote the lipid deposition of goat intramuscular precursor adipocytes by promoting the expression of genes related to cell lipid production and reducing the expression of genes related to lipolysis, which provided an experimental reference for further revealing the role and molecular mechanism of FATP1 gene in regulating lipid metabolism.

Investigation and Analysis of Nucleic Acid Detection Results of Viral Viruses in Large-Scale Goose Farms

SHAO Zhen, DIAO YouXiang

Scientia Agricultura Sinica. 2023, 56(10): 2021-2034. doi:10.3864/j.issn.0578-1752.2023.10.016

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【Objective】The author’s research group has long investigated the nucleic acid detection rate of common viral diseases in scaled goose farms in various provinces and cities in China, and found that many large-scaled goose farms in China could detect multiple viral nucleic acids at the same time, and this phenomenon was very common. In view of the lack of relevant data on the simultaneous detection of multiple viral diseases in large-scale goose farms in China, the purpose of this paper was to understand and analyze the simultaneous detection of nucleic acids of multiple viral diseases in large-scaled goose farms, so as to provide the theoretical guidance and scientific basis for the prevention and control of viral diseases in scaled goose farms.【Method】From May 2021 to October 2021, 737 diseased materials were collected from 47 scaled goose farms in Shandong, Heilongjiang, Sichuan, Jilin, Guangxi, Henan, Anhui, Liaoning, Hebei, Guizhou, Hunan and Inner Mongolia. These samples were detected for Muscovy duck reovirus (MDRV), duck reovirus (DRV), goose astroviruses (GAstV), fowl adenovirus (FAdV), reticuloendotheliosis virus (REV), Newcastle disease (NDV), goose parvovirus (GPV), goose circovirus (GoCV), goose hemorrhagic polyomavirus (GHPV), Tembusu virus (TMUV) and H9 subtype avian influenza virus (H9-AIV) by ordinary PCR and RT-PCR. The liver, spleen, lung and kidney were dissected from each sample, and total RNAs were extracted by Trizol method; a strand of cDNA was synthesized by reverse transcription with total RNA as the template, and then the complete cDNA was obtained by continuous amplification with cDNA as the template. Using these cDNA as template, the specific primers of MDRV, DRV, GAstV, FAdV, REV, NDV, GPV, GoCV, GHPV, TMUV and H9-AIV were used to amplify the target fragment by ordinary PCR reaction. All amplified fragments were analyzed by agarose gel electrophoresis, and some positive samples were sequenced. The obtained sequencing results were compared with the corresponding virus gene sequences published on GenBank, and the phylogenetic tree was drawn by Neighbor-Joining (N-J) method in MEGA 6.0 software for analysis.【Result】MDRV, DRV, GAstV, FAdV, REV, NDV, GPV, GoCV, GHPV, TMUV and H9-AIV showed that the detection rate of GAstV was the highest, with the value of 58.21%; the detection rates of REV and NDV were the lowest, with the value of 1.36% and 1.50%, respectively. A variety of viral nucleic acids could be detected simultaneously in geese to varying degrees, especially in two or three viral nucleic acids, accounting for 67.44% of the total samples. In the simultaneous detection rate of nucleic acids of the two viruses, GAstV and GoCV accounted for the largest proportion, which was 18.44%; MDRV, GAstV and GPV accounted for 36.28% of the simultaneous detection rates of the three virus nucleic acids.【Conclusion】It was further confirmed that multiple viral nucleic acids could be detected simultaneously in large-scaled goose farms in China. It was speculated that this might be one of the important reasons for the complexity of viral diseases and the difficulty of prevention and control in large-scaled goose farms in China.

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