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    16 June 2019, Volume 52 Issue 12
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Expression Profiling and Functional Characterization of Rice Transcription Factor OsWRKY68
    Yue CHEN, TianXingZi WANG, Shuo YANG, Tong ZHANG, JinJiao MA, GaoWei YAN, YuQing LIU, Yan ZHOU, JiaNan SHI, JinPing LAN, Jian WEI, ShiJuan DOU, LiJuan LIU, Ming YANG, LiYun LI, GuoZhen LIU
    Scientia Agricultura Sinica. 2019, 52(12):  2021-2032.  doi:10.3864/j.issn.0578-1752.2019.12.001
    Abstract ( 698 )   HTML ( 100 )   PDF (1035KB) ( 467 )   Save
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    【Objective】 There are nearly 100 WRKY transcription factor members in rice genome, many of them are involved in plant growth and development, biotic and abiotic stress responses. Molecular biology & bioinformatics lab identified that the expression of OsWRKY68 protein was induced after inoculation with Xanthomonas oryzae pv. oryzae (Xoo) in rice. The aim of this study is attempt to further explore the function of OsWRKY68. 【Method】Rice TP309 samples of different tissues at different developmental stages, including germination, seedling, tillering, booting and flowering stages of root, stem, leaf, sheath, cushion, panicle, anther, husk, seed, abiotic stress (4℃, 44℃, 48℃, submerge, NaCl, PEG, constant light, constant dark) and hormone treatments (abscisic acid, methyl jasmonate, salicylic acid, ethephon) were collected. Total protein were extracted and analyzed by Western blot (WB) systematically using OsWRKY68-specific antibody. The expression patterns of OsWRKY68 protein isolated from different tissues at different developmental stages, and tissues obtained from abiotic stresses and hormone treatments were investigated. RNA interfering vector was constructed and transformed to wildtype TP309 rice variety via Agrobacterium tumefaciens strategy. Identification of transgenic plants were carried out by PCR and WB. The phenotype of OsWRKY68 RNAi transgenic plants were monitored and plant height, tiller number, spike length, spikelet number and seed-setting rate were measured.【Result】By comparing the abundance of OsWRKY68 protein in different tissues, it was found that OsWRKY68 protein was expressed in a constitutive way during the normal growth and development of rice, the abundance of OsWRKY68 protein expressed among different tissues were not varied too much. However, different levels of OsWRKY68 were observed. The expression level of OsWRKY68 in anthers at flowering stage was higher than that in mature panicles, panicle axis and husk. It was not expressed in sheaths at tillering and booting stages, but it was expressed in sheaths at flowering stage. In panicles, the abundance of OsWRKY68 was decreased gradually along with the growth of the young panicle. By investigating the expression patterns of OsWRKY68 protein under abiotic stress and hormone treatments, it was found that the abundance of OsWRKY68 protein decreased steadily under salt stress. The expression of OsWRKY68 protein increased steadily at constant light treatment, a specific band (designated as OsWRKY68 +) with higher molecular weight appeared at three days and enhanced in the following timepoints. After methyl jasmonate (MeJA) and ethephon (ET) treatments, OsWRKY68 + band appeared also and its intensity increased as the treatments continues. Four homozygous OsWRKY68 RNAi transgenic lines (Y316, Y317, Y326 and Y337) were checked by PCR and WB analyses and verified at T3 generation. The abundance of OsWRKY68 protein in RNAi transgenic plants was lower than that in wildtype TP309. Phenotypic investigation revealed significant reduction in plant height, tiller number and seed setting rate in transgenic plants.【Conclusion】Rice OsWRKY68 protein plays an important role in the process of normal growth and development of rice. Knocking down the abundance of OsWRKY68 protein via RNAi affected the normal growth of rice. In addition, the data of expression patterns suggested that the function of OsWRKY68 protein may be involved with salt stress, light, MeJA and ethene-mediated signal transduction pathways.

    QTL Mapping and Genetic Analysis of a Gene with High Resistance to Southern Corn Rust
    WANG BingWei, QIN JiaMing, SHI ChengQiao, ZHENG JiaXing, QIN YongAi, HUANG AnXia
    Scientia Agricultura Sinica. 2019, 52(12):  2033-2041.  doi:10.3864/j.issn.0578-1752.2019.12.002
    Abstract ( 492 )   HTML ( 26 )   PDF (854KB) ( 446 )   Save
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    【Objective】This study aimed at constructing regional genetic map and positioning the resistant gene to Southern Corn Rust by using one of the 8 F2 populations, deriving from crosses of the highly resistant tropical maize line S313 with 4 highly susceptible inbred lines to Southern Corn Rust, in 3 seasons of two years. In addition, a major QTL for Southern Corn Rust resistance was fine-mapped by applying new-developed molecular markers to a larger mapping population.【Method】Total 8 F2 populations were planted in 3 consecutive seasons of 2016 and 2017, all of which received triple inoculation. Individual F2 plants grown in the field were scored for resistance to southern rust using a nine-point relative scale at late growth stage. Finally, the segregation ratio of resistant to susceptible plants for each F2 population was investigated by phenotypic identification. A 56K-SNP chip was used for screening polymorphisms between the two parents, among which 192 SNP markers were picked for genotyping the 30 high resistant and 30 susceptible plants in an F2 population of S313×PHW52. Nineteen newly developed SNP markers on the short arm of chromosome 10 were used for genotyping the whole F2 population and constructing a regional genetic map. The resistant QTL mapping was proceeded by the combination of genetic map and phenotypic identification. The genotyping to second population with 10 newly developed SNP markers within the initial mapping region was performed, and the interval was delimited according to the number of exchanged plants. According to the information of maize B73 genome, all genes in corresponding interval were listed. By the gene functional annotation information, the candidate genes were determined.【Result】 By Chi-square test, the ratio of resistant plants to susceptible plants in these 8 F2 populations was all statistically a 3:1 ratio, which indicated that the resistance to Southern Corn Rust of S313 was controlled by a main effect QTL. A total of 16426 polymorphic markers between the two parents were screened out from the 56K-SNP chip. A resistance-linked marker Affx-90241059 was identified by the linkage analysis of 30 resistant and 30 susceptible plants with 192 markers of uniform distribution across chromosomes. A regional genetic map with total distance of 31.8cM and average distance 1.77 cM was constructed by using 19 SNPs. By the composite interval mapping method, the main effect QTL was located within the interval between marker Affx-91298359 and marker Affx-91182449, which size was 2M approximately. Furtherly, the interval was delimited to the size of 474K by using a larger F2 population and 10 SNP markers. There are 63 genes in the 474k interval of B73 reference genome, among which, gene LOC103640657, LOC100191493 and LOC103640673 encode plant disease resistance-related proteins. Thus, these three genes were listed as candidate genes resistant to Southern Corn Rust of tropical maize inbred line S313. 【Conclusion】 By field phenotypic identification, the ratio of resistant plants to susceptible plants in all F2 populations was determined as 3:1, which indicated that the resistance to Southern Corn Rust of S313 was under controlled by a main effect QTL. By the methods of population construction and molecular marker, the main effect QTL of S313 was located in the interval of about 0.47M size on the short arm of maize chromosome 10. There are 3 candidate genes related to Southern Corn Rust in this region.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Impacts of Soil Tillage Techniques on Seedling Quality, Root Function and Grain Weight in Wheat
    SHEN GuanYu, YANG XiWen, ZHOU SuMei, MEI JingJing, CHEN Xu, PENG HongYang, JIANG Xiang, HE DeXian
    Scientia Agricultura Sinica. 2019, 52(12):  2042-2055.  doi:10.3864/j.issn.0578-1752.2019.12.003
    Abstract ( 588 )   HTML ( 45 )   PDF (446KB) ( 404 )   Save
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    【Objective】 In view of the prominent problem that corn straw to the field seriously affects the quality of wheat (Triticum aestivum L.) seedlings under the condition of double cropping per year of Huang-Huai Plain, appropriate soil tillage techniques were explored to improve quality of wheat seedlings and ultimately to increase wheat yield. 【Method】The experiment was carried out at Huanggang village, Xindian town, Xinzheng city, Henan province for 2 consecutive years (from 2016 to 2018). By using a randomized block design, 8 treatments were carried out by combining three factors of plowing, harrow and compacting: deep depth tillage + rotary tillage (DT + RT; DT: 30 cm, RT: 15 cm), deep tillage + harrow (DT + H), deep depth tillage + rotary tillage + compacting (DT + RT + C), rotary tillage (RT), rotary tillage + harrow (RT + H), rotary tillage + compacting (RT + C), and rotary tillage + harrow +compacting (RT + H + C). Emergence rate and seedling quality of wheat were investigated, root characteristics were investigated during wintering stage, re-growing stage, jointing stage, heading stage, grain filling stage, dough stage, and grain characters, yield and its components were analyzed during the filling period, respectively. 【Result】20 days sowing, quality of seedlings in different soil tillage treatments was significantly different. In rotary tillage treatments, emergence uniformity was higher than that in deep tillage treatments, while in deep tillage method, seedling emergence rate, basic seedlings and plant height were higher than those in rotary tillage method. Under the same plowing and compacting, emergence rate in harrow treatment emergence was 1.0%-5.7%; Under the same plowing and harrow, emergence rate in compacting treatment emergence was 0.06%-8.3%. At the same time, after deep depth tillage, seedling deficiency and wedging were rarely seen. Cumulative length of seedling deficiency and wedging under RT treatment was the highest, and the average length of the two years was 55 cm. From the wintering to dough stage, root activity in different soil tillage treatments showed a "high-low-high-low" trend, and it was the highest in treatment “DT + H + C”. In wintering and at jointing stage, root activity was increased after treated with compacting and harrow compared with that in treatments with neither compacting nor harrow. Secondary roots per plant reached the maximum in DT + H + C treatment at heading stage with the highest values were 45.2 and 40.2 in 2017 and 2018, respectively. After treated with harrow, secondary roots per plant were increased by 14.8% compared with those in treatments without harrow. After compacted treatment, secondary roots per plant were increased by 12.2% compared with those in treatments without compacting. Grain filling rate in DT + H + C treatment was significantly higher than that under other treatments during 5-10 days after anthesis, and reached its peak at 20 days after anthesis. Grain filling rate of DT + H + C treatment was higher than that under other treatments, while grain filling rate in DT + H + C treatment was higher than that under other treatments, and grain filling rate under DT + H + C treatment was 19.4% higher than that under other treatments (α=0.05). During grain filling stage, 1000-grain weight increased the most at 0-15 days after anthesis; Under DT + H + C treatments, it was increased the most; Under DT + RT treatments, it was increased the least; under DT + H + C treatments, it increased the most at 25-30 days after anthesis, which was 20.8% higher than that when treated by RT. According to the effects of different tillage techniques on grain yield and its components, the highest grain yield was obtained under DT + H + C treatment. Ears per unit area, kernels per ear and 1000-grain weight were not regular under harrow milling and repressing treatment, and grain yield increased obviously, with a range of 1.4%—12.2%. Economic benefits, in RT + H, RT + H + C, RT + C, DT + H + C, or DT + H treatment were all higher than those in RT treatment compared with the previous farming methods in the local area. Among them, the highest economic benefits was produced under DT + H + C treatment, and the two-year average was 12.3% higher than that under RT treatment【Conclusion】Under the current condition of two cropping systems in the Huang-Huai plain, different soil tillage techniques affected seedling quality, rotation tillage was beneficial to uniformity of seedling emergence, while deep tillage was beneficial to increasing seedling emergence rate, seedling evenness and plant height, laying a foundation for the formation of strong seedlings before wintering. The deep tillage deepened soil surface layer, which was beneficial to rooting, increased secondary roots and root activity in soil surface layer, and indirectly affected grain yield. Considering root system growth, grain filling rate, grain weight formation and yield performance, it was concluded that DT + H + C soil tillage technique was the best choice in Huang-Huai area.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Parameter Optimization for the Simulation of Leaf Area Index of Dryland Wheat with the APSIM Model
    NIE ZhiGang,LI Guang,WANG Jun,MA WeiWei,LUO CuiPing,DONG LiXia,LU YuLan
    Scientia Agricultura Sinica. 2019, 52(12):  2056-2068.  doi:10.3864/j.issn.0578-1752.2019.12.004
    Abstract ( 446 )   HTML ( 16 )   PDF (814KB) ( 317 )   Save
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    【Objective】The effective application of the model depends on the fast and accurate estimation of parameters. The current problems in the calibration of crop growth model parameters include large data volume, long time consumption, lack of precision, and low efficiency. The study tried to solve the problems. 【Method】Based on the field experimental data of two experimental sites (Mazichuan village, Lijiabaotown and Anjiagou village, Fengxiangtown) in Andingdistrict, Dingxi city in multiple years (2002-2005 and 2015-2017) and the meteorological data in Andingdistrict, Dingxicity from 1971 to 2017, the parameters related to dryland wheat leaf area index (LAI) in the APSIM (agricultural production systems simulator) model were optimized with the intelligent iteration search principle of shuffled frog leaping algorithm (SFLA) and tested by the correlation analysis method. 【Result】The biological evolution learning strategy of local depth search within sub-group and global information communication between sub-group in frog population, which was relatively independent and coordinated, was used to effectively improve the speed of calculation and realize the fast and accurate estimation of the parameters related to dryland wheat LAI in the APSIM model. The related parameters mainly included: The required thermal time interval for node appearance on the main stem, the initial node number at emergence, the initial leaf number at emergence, the initial leaf area index at emergence, the growing node number, and the maximum specific leaf area. LAI was respectively simulated by using the parameters based on the trial and error method and based on SFLA. After parameter optimization, the root mean square error (RMSE) between simulated and measured wheat LAI reduced from 0.069 to 0.027, the normalized root mean square error (NRMSE) decreased from 8.09% to 4.56%, and the model effective index (ME) increased from 0.979 to 0.993. 【Conclusion】Compared with the trial and error method, which was usually used in the calibration of APSIM model, the intelligent iterative behavior with spontaneous learning characteristics based on the SFLA could realize automatic calibration of the parameters and improve the efficiency. The parameters estimated based on the SFLA could remarkably improve the simulation accuracy of wheat LAI. The application of SFLA was effective in calibrating crop models involving complex eco-physiological processes, and it could provide an effective parameter optimization method for improving the disadvantages in the model parameter calibration process include large data volume, long time consumption, lack of precision, and low efficiency.

    PLANT PROTECTION
    Establishment and Application of a Triplex PCR Detection System for Vegetable Soil-Borne Pathogens
    LIU RuiChi,CHENG YouPu,CHAI ALi,SHI YanXia,XIE XueWen, PATIGULI,LI BaoJu
    Scientia Agricultura Sinica. 2019, 52(12):  2069-2078.  doi:10.3864/j.issn.0578-1752.2019.12.005
    Abstract ( 418 )   HTML ( 24 )   PDF (576KB) ( 208 )   Save
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    【Objective】The objective of this study is to establish a triplex PCR system for the detection of Pythium aphanidermatum, Fusarium oxysporum and Verticillium dahliae, and to provide technology and method for early diagnosis and identification of soil-borne diseases of vegetables.【Method】Three sets of specific primers were selected to analyze the influencing factors of triplex PCR and the primer concentration, annealing temperature, amplification cycles and extension time of PCR reaction were optimized. The best triplex PCR detection system for vegetable soil-borne pathogenic fungi was established and the sensitivity of the system was detected. In order to test the stability of the system, 2×Taq Master PCR Mix (Beijing Biomed Co. Ltd) and TaKaRa Taq enzyme (TaKaRa Biotechnology Dalian Co. Ltd) were used by C1000 Touch TM (Thermo Fisher Scientific Co. Ltd) and Aeris TM type (Esco Micro Pte Ltd) thermal cycle meter for amplification. The triplex PCR and pathogen isolation and detection of 35 disease samples and 149 soil samples collected in the field were carried out to determine the applicability of the triplex PCR detection system.【Result】In the triplex PCR detection system, the specific target fragments of P. aphanidermatum, F. oxysporum and V. dahliae with the length of 163, 328 and 530 bp could be amplified by AsAPH2B/AsPyF, FOF1/FOR1, VActF/VActR, respectively. The 25 μL reaction system contained 0.12 μmol·L -1 AsAPH2B/AsPyF, 0.16 μmol·L -1 FOF1/FOR1, 0.24 μmol·L -1 VActF/VActR, 2×Taq Master PCR Mix 12.5 μL. The annealing temperature was 60.8℃ and the number of cycles was 35. The detection limitation was 10 -1ng·μL -1for pure culture of pathogens. For artificially infected substrate, the detection limitations were 10 5, 10 6 spores/g and 10 -2 mg hyphae/g for V. dahliae, F. oxysporum and P. aphanidermatum, respectively. The results were consistent of 2×Taq Master PCR Mix and TaKaRa Taq enzyme, which were amplified by C1000 Touch TM and Aeris TM type thermal cycle meter. It meant that the triplex PCR system was stable. The infected tissues and soil samples collected in the field were detected, and 25 tissues and 71 soil samples were detected for carrying pathogen, which were consistent with the results of isolation and culture.【Conclusion】The triplex PCR detection system established in this study has the characteristics of high sensitivity, stability and reproducibility, and can be used for rapid and accurate detection of P. aphanidermatum, F. oxysporum and V. dahliae in the infected plant and the surrounding soil. It provides an effective technical means for early prevention and epidemic monitoring of vegetable soil-borne diseases.

    Molecular Regulation of Trichoderma harzianum ACCC32527 Response to NaCl Based on Transcriptome and Metabolome Analysis
    XIANG Jie, CHEN JingShi, XIA XinXin, LIU Kuai, LI ShiGui, GU JinGang
    Scientia Agricultura Sinica. 2019, 52(12):  2079-2091.  doi:10.3864/j.issn.0578-1752.2019.12.006
    Abstract ( 348 )   HTML ( 12 )   PDF (6641KB) ( 277 )   Save
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    【Objective】The objective of this study is to mine the key functional differentially expressed genes (DEGs) and secondary metabolites by analyzing the data of Trichoderma harzianum ACCC32527 differential transcriptomes and metabolomes under NaCl stress.【Method】The transcriptomes and metabolomes of ACCC32527 with the treatment of 0 (T1), 0.4 (T2), 0.6 (T3) mol?L -1 NaCl were performed by RNA-seq and GC-TOF-MS, respectively, and the annotation, screening and classification of DEGs (|log2fold change|>1 & FDR<0.01) and secondary metabolites (P-value≤0.05 & VIP>1) were completed by related softwares and databases (GO, COG and KEGG pathway). Finally, RT-qPCR was conducted to validate RNA-seq data.【Result】In this study, after NaCl stress treatments, the growth of ACCC32527 was inhibited and the reproductive rate was significantly decreased, 637 and 1 570 DEGs were screened from the T1 vs T2 and T1 vs T3 comparison groups, respectively. Trend analysis result showed that 1 921 DEGs were assigned to 16 significant expression patterns, including 9 up-regulated trends (950 genes), 3 down-regulated trends (662 genes) and 4 irregular trends (309 genes). GO enrichment analysis result showed that each tread was shared by catalytic activity, binding, organelle, cell membrane part, cell membrane, cell part, cell, single-organism process and metabolic process, and accounted for 61%-94%. Among them, the classification with great difference in gene proportion before and after treatment was catalytic activity. The enrichment analysis of KEGG metabolic pathway displayed that the classification and numbers of pathways treated with different NaCl concentrations were significantly different, there were 225 and 535 DEGs enriched in 20 KEGG pathways, respectively, including metabolic pathways, biosynthesis of antibiotics, biosynthesis of secondary metabolites and so on. Among them, 12 and 18 related genes of ribosome biosynthesis pathway were suppressed under T2 and T3 treatments, respectively. A total of 73 genes were screened from the DEGs including ROS scavenging, ion transport and cell wall structure, and most of them were up-regulated. In addition, intracellular small molecule metabolites showed significant change in response to NaCl stress, and there were 30 metabolites with increased accumulation, including amino acids and their derivatives, sugars and their derivatives and alcohols, while 53 metabolites were reduced including fatty acids and organic acids. Among them, glycerol, a known important osmoregulation substance of fungi, was increased under T3 treatment, which may be involved in the process of cell osmotic adjustment. RT-qPCR verified the expression changes of 7 DEGs, which were consistent with the RNA-seq analysis, indicating the reliability of transcriptome sequencing results.【Conclusion】Under NaCl stress, a large number of genes and secondary metabolites of T. harzianum ACCC32527 were changed, which were related to NaCl stress regulation. These processes act together to reduce the toxic effect to the cells, and the results can provide important information for the study of salt-tolerant mechanism of Trichoderma spp..

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Analysis of Fertilizer Application and Its Reduction Potential in Paddy Fields of Heilongjiang Province
    PENG XianLong,WANG Wei,ZHOU Na,LIU HaiYang,LI PengFei,LIU ZhiLei,YU CaiLian
    Scientia Agricultura Sinica. 2019, 52(12):  2092-2100.  doi:10.3864/j.issn.0578-1752.2019.12.007
    Abstract ( 364 )   HTML ( 24 )   PDF (472KB) ( 289 )   Save
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    【Objective】Heilongjiang has a paddy field of more than 3.2 million hectares, it is the largest province in China with rice fields. In the past ten years, rice production in Heilongjiang Province has been hovering at 7 000 kg·hm -2, while it was the province with the lowest amount of fertilizer in paddy fields in China. Under the background of “fertilizer zero increase”, whether is there potential for fertilizer saving in Heilongjiang Province remains to be studied. 【Method】We investigated paddy field fertilization across different farms in the main rice producing areas in Heilongjiang Province. In 2005, the survey areas were Wuchang, Fangzheng, Mulan, Ning'an, Qing'an, Tieli, Shangzhi and Acheng; in 2008, the survey areas were Mishan, Hulin, Qing'an, Wuchang, Ning'an, Fangzheng, Luobei, Huachuan, Fujin and Shangzhi; the survey areas in 2015 were Wuchang, Fangzheng, Ning'an, Hulin and Qing'an. From each county, a random township was selected, and 2 or 3 villages were randomly selected from each township, then 10 households were surveyed in each village. From 2009 to 2010, more than 80,000 soil samples from the main rice producing areas in Heilongjiang Province were collected, and the phosphorus and potassium nutrient contents in 0-20 cm soil layer were determined. The nitrogen application rate was estimated through theoretical appreciate application rate method. Based on the crop nutrient demand and the soil nutrient status of the paddy field, phosphorus and potassium application rate was evaluated. Fertilizer reduction potential was analyzed in Heilongjiang Province.【Result】According to the research data, the average rice yields in 2005, 2008 and 2015 were 6 427, 7 593 and 7 142 kg·hm -2, respectively, and the average annual output was 7 104 kg·hm -2. The crop yield differed between farms with a range of nearly 5 000 kg·hm -2. The average amount of N, P2O5 and K2O in paddy fields were 141.0, 56.6 and 51.6 kg·hm -2, respectively, and the difference in the amount of NPK fertilizers was more than 300 kg·hm -2. Therefore, the results showed that fertilization variation among farmers was large, so the problem of blind fertilization was prominent. The content of available phosphorus and available potassium in the paddy soil of Heilongjiang Province were about 26 mg·kg -1 and 138 mg·kg -1, respectively. The variation of available phosphorus in soil exceeded 40%, and soil fertility varied greatly among different regions. High level phosphorus and potassium accounted for above 70% samples. To reach rice production levels of 7 500 kg·hm -2, the theoretical suitable nitrogen application rate was 105 kg·hm -2. Only 20% of farmers achieved high yield and high nitrogen efficiency, and 70% of the farmers had fertilizer-saving potential with nitrogen reduction of 26%. Through fertilizer saving, the grain produced per kilogram of nitrogen could be increased from 50 kg to 70 kg. According to the current yield and soil nutrient status, the appropriate amount of phosphate and potassium fertilizer in paddy fields was only 41.6 and 35.9 kg·hm -2, which could be reduced by about 30%. Among the surveyed households, farmers whose fields had the potential for phosphorus and potassium-saving accounted for 71% and 72% of the total, respectively, among which the low-yield and low-efficiency farmers accounted for 30% of the total farmers, where the fertilizer-saving potential was the largest.【Conclusion】Although Heilongjiang was the province with the lowest fertilization rate in China, about 70% of farmers were high-yield but low efficiency, or low-yield and low efficiency, so the problem of excessive fertilization was prominent, and the fertilizer-saving potential was more than 20% in Heilongjiang Province.

    Comparative Study on CH4Emission from Ratoon Rice and Double-Cropping Rice Fields
    ZHANG Lang,XU HuaQin,LI LinLin,CHEN YuanWei,ZHENG HuaBing,TANG QiYuan,TANG JianWu
    Scientia Agricultura Sinica. 2019, 52(12):  2101-2113.  doi:10.3864/j.issn.0578-1752.2019.12.008
    Abstract ( 447 )   HTML ( 14 )   PDF (1159KB) ( 255 )   Save
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    【Objective】In order to explore the ecologically sustainable rice planting model, the yield potential and CH4 emission characteristics of traditional double-cropping rice and emerging ratoon rice in the middle and lower reaches of the Yangtze River were compared, so as to provide a scientific basis for selecting a green and ecologically sustainable rice farming model.【Method】In 2017-2018, relying on the research base of Hongshuo Eco-Agricultural Machinery Company in Datonghu District, Yiyang City, Hunan Province, two models of double-cropping rice and ratoon rice were set up, and the yield potential, CH4 emission dynamics and CH4 seasonal cumulative emission were compared and analyzed to evaluate unit yield CH4 emissions from paddy fields.【Result】During the experiment, from the aspect of yield, the yield of early rice was 7.37 t·hm -2, and the yield of main crop was 8.84 t·hm -2. Main crop yield increased by 19.95% compared with early rice; the late rice yield was 6.82 t·hm -2, and the ratoon crop was 3.39 t·hm -2. Compared with the late rice, the ratoon crop yield was reduced by 50.29%. In the two crops, the total yield of double-cropping rice was 14.19 t·hm -2, and the total yield of ratoon rice was 12.22 t·hm -2; in terms of CH4 emission dynamics, double-cropping rice was highly polluted in the tillering and full-heading. In addition to strong emissions in the tillering and full-heading, the ratoon rice also appeared small peak when applying budding fertilizer. However, the emission range of the overall double-cropping rice (-0.06 to 1.30 μmol·m -2·s -1) was higher than that of the ratoon rice (- 0.01 to 0.70 μmol·m -2·s -1); from the seasonal cumulative emission of CH4 in paddy fields, the cumulative emission of CH4 in double-cropping rice was higher than that in ratoon rice. The cumulative emission range of main crop was from 23.90 to 266.59 kg·hm -2, and the ratoon crop was from 0 to 4.61 kg·hm -2. The cumulative emission range of early rice was from 35.57 to 251.29 kg·hm -2, and the late rice was from10.74 to 321.59 kg·hm -2. CH4 seasonal cumulative emissions of double-cropping rice showed: A-B stage (two leaves-one heart to late-tillering) > B-C stage (late-tillering to full-heading) > C-D stage (full-heading to mature), and cumulative emissions of double-cropping rice during the whole growth period was up to 922.35 kg·hm -2. The cumulative emission of methane from ratoon rice was B-C stage>A-B stage>C-D stage, and the cumulative emission of methane during the whole growth period was 609.74 kg·hm -2. Compared with the double-cropping rice control, ratoon rice methane cumulative emissions decreased by 33.89%. Finally, by evaluating the unit yield methane emissions, the methane emission per unit yield of early rice was0.069 kg·kg -1, and the methane emission per unit yield of main crop was 0.062 kg·kg -1, which was 10.14% lower than that of early rice. The methane emission per unit yield of late rice was 0.061 kg·kg -1, the methane emission per unit of ratoon crop was0.018 kg·kg -1, and the ratoon crop was reduced by 70.49%, compared with the late rice. In the two crops, the methane emission per unit yield of double-cropping rice was 0.065 kg·kg -1, and the methane emission per unit yield of ratoon rice was 0.050 kg·kg -1, which was 23.08% lower than that of double-cropping rice.【Conclusion】Therefore, in terms of methane emissions per unit yield, expanding the cultivation of ratoon rice was a good strategy in the main producing areas of the double-cropping rice in the middle and lower reaches of the Yangtze River.

    Optimal Protected Area Selection: Based on Multiple Attribute Decision Making Method and Ecosystem Service Research ——Illustrated by Guanzhong-Tianshui Economic Region Section of the Weihe River Basin
    ZHANG YuMeng,LI Jing,ZENG Li,YANG XiaoNan,LIU JingYa,ZHOU ZiXiang
    Scientia Agricultura Sinica. 2019, 52(12):  2114-2127.  doi:10.3864/j.issn.0578-1752.2019.12.009
    Abstract ( 360 )   HTML ( 19 )   PDF (5322KB) ( 308 )   Save
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    【Objective】 In present study, based on the calculation of biodiversity, carbon sequestration, water production and cultural ecosystem services, multi-attribute decision-making method is applied to select the optimal protected area of the study area. The work can provides a theoretical basis for the maintenance of ecological balance and ecological construction in the study area. 【Method】Our research is illustrated by the Guanzhong-Tianshui Economic Zone (Guantian Section) of Weihe River Basin, biodiversity and carbon sequestration services are calculated by InVEST model, SWAT model is used to measure water yield services and divide sub-basins, cultural services are quantified by SoLVES model. On this basis, 11 scenarios are established by using OWA (Ordered Weighted Averaged) multi-attribute decision making method, and the priority protected areas are screened out. 【Result】 (1) The distribution of water yield in the study area was uneven on the sub-basin scale. On the whole, the water yield in the eastern region is the highest, and that in the central region is smaller than that in the western region, and the average value is about 95 mm. The range of carbon sequestration on the sub-basin scale is between 5.041 t·hm -2 and 10.284 t·hm -2. Sub-basins with higher carbon sequestration per unit area are mostly located in Qinling area, and the sub-basins located in Guanzhong Plain are urban agglomeration area, and the regional carbon sequestration with dense construction land is very limited. Biodiversity is mainly measured by habitat quality index, the range of habitat quality in the study area is between 0.222 and 0.921. The vegetation cover is insufficient in Guanzhong Plain area, frequent human activities and urban development lead to dense distribution of threat factors. Therefore, the habitat quality is relatively low with 0.22 in Guanzhong Plain area. The region of Qinling Mountains, with high vegetation cover, is far from the habitat threat factor, its ecological suitability is relatively high and the biodiversity is maintained well. The total index of cultural value in the study area is between 1.298 and 5.667. The aesthetic and spiritual values are concentrated in forest parks and Qinling Mountains, while urban areas are rich in historical heritage and entertainment facilities , as a consequence, the urban area has the highest value of cultural services. (2) By setting up different risk values and weights to calculate OWA operator, 11 risk scenarios are obtained and their protection efficiency are calculated. Finally, risk scenario 6 is selected as the optimal protected area with highest trade-off value, its protection efficiency is about 1.499. The range of risk scenario 6 is mainly located in the southern Qinling area and the junction part of Tianshui City and Baoji City. 【Conclusion】this study analyzes and evaluates ecosystem services in the study area by combining natural and human factors. The OWA operator is used to balance the trade-offs between different ecosystem services and to select the optimal protected areas, which provides a reference for improving ecosystem services in the study area.

    HORTICULTURE
    Evaluation Indexes for Blueberry Quality
    ZHANG Jia, NIE JiYun, ZHANG Hui, LI Jing, LI Ye
    Scientia Agricultura Sinica. 2019, 52(12):  2128-2139.  doi:10.3864/j.issn.0578-1752.2019.12.010
    Abstract ( 461 )   HTML ( 10 )   PDF (490KB) ( 317 )   Save
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    【Objective】 The aim of this study was to explore the relationship among the indexes of blueberry quality and to establish their scientific grading standards, so as to provide a theoretical reference for reasonable evaluation of blueberry quality.【Method】Seven indexes (fruit weight, fruit firmness, titratable acidity, total soluble solid, total soluble solid/titratable acidity, fruit shape and vitamin C) of samples were determined, and the relationship among indexes were probed with correlation and regression analysis. Indexes were graded by probability grading, and typical indexes were identified by principal component analysis and systematic cluster analysis. The weight of indicator scores was identified by analytic hierarchy process. 【Result】 The dispersion degree varied greatly among blueberry quality indexes with the variable coefficient of 67.12% (total soluble solid/titratable), 45.46% (titratable acidity), 35.28% (fruit weight), 23.48% (vitamin C), 18.23% (total soluble solid), 18.05% (fruit firmness), and 6.05% (fruit shape). Both titratable acidity and vitamin C distributed normally, with probability values above 0.05. The distribution of fruit weight could be considered as normal with the probability value of 0.0494 (close to 0.05). If some extreme values were removed, other three indexes (fruit shape, fruit firmness, and total soluble solid) also distributed normally. Titratable acidity had a significant negative correlation with total soluble solid/titratable acidity, with correlation coefficient of -0.81742. Total soluble solid/titratable acidity had a significant power function concerning titratable acidity, and the coefficient of determination was 0.9005. From the evaluated seven indexes, four indexes (titratable acidity, total soluble solid, vitamin C and fruit firmness) were screened as evaluation indexes of blueberry quality, which represented sour index, sweet index, nutritional index and texture index respectively. These four indexes could be divided into 5 grades (lower, low, medium, high, and higher) with normal distribution, and the scoring standards of them were also established. By using these four selected indexes, blueberries from different regions were divided into three groups, including superior, medium and inferior. 【Conclusion】Blueberry quality could be evaluated by 4 indexes, including total soluble solid, titratable acidity, vitamin C and fruit firmness. The established scoring standards of four indexes could be effective in evaluation and classification of blueberry quality.

    Effects of Salt Stress on Seedlings Growth, Photosynthesis and Chlorophyll Fluorescence of Two Species of Artocarpus
    SU LanXi,BAI TingYu,YU Huan,WU Gang,TAN LeHe
    Scientia Agricultura Sinica. 2019, 52(12):  2140-2150.  doi:10.3864/j.issn.0578-1752.2019.12.011
    Abstract ( 488 )   HTML ( 33 )   PDF (1188KB) ( 251 )   Save
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    【Objective】The effects of salt stress on the growth, photosynthesis and chlorophyll fluorescence of the seedlings of two Artocarpus species were studied in order to provide a reference for the utilization of tropical saline-alkali land and the development of characteristic agriculture on islands. 【Method】 Jackfruit and breadfruit, two Artocarpus varieties, were selected and cultivated in sea sand. The sea water solution was applied at 3-leaf full expanding stage and the salt concentration was 0, 3.4, 10.2, 17.0 and 23.8 g?kg -1, named as T0, T1, T2, T3 and T4, respectively. The variation of growth, photosynthetic and chlorophyll fluorescence of jackfruit and breadfruit seedlings under salt stress were investigated. 【Result】 Salt stress significantly inhibited the growth, chlorophyll content and biomass accumulation of jackfruit, and the inhibition effect on breadfruit was less than that of jackfruit. The parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci) and transpiration rate (Tr) of jackfruit, were decreased under salt stress, while the stomatal inhibition (Ls) was increased. The parameters of breadfruit changed less than that of jackfruit. High salt stress (salt concentration>17.0 g?kg -1) significantly inhibited the photochemical quenching coefficient (qP), PSII activity (Fv/Fo), potential maximum light energy conversion efficiency (Fv/Fm), actual light energy conversion efficiency (ΦPSII), relative electron transfer efficiency (rETR) of jackfruit, and significantly decreased qP, non-photochemical quenching coefficient (NPQ), ΦPSII and rETR of breadfruit. There was a significant positive correlation between chlorophyll content and photosynthetic parameters. The proportion of salt stress, chlorophyll content, net photosynthetic rate and their mutual interaction to explain the change of biomass was 35.6%. 【Conclusion】 Salt stress significantly inhibited the growth, chlorophyll content, photosynthetic parameters and biomass of jackfruit seedlings, while the photosynthetic parameters and biomass of breadfruit were sensitive to high salt stress. The changes of salt stress, chlorophyll content and net photosynthetic rate were closely related to the biomass variation of two Artocarpus varieties.

    FOOD SCIENCE AND ENGINEERING
    Quality Evaluation System Established for Pear Processed by Freezing in China
    WANG Yang, JIA XiaoHui, WANG WenHui, TONG Wei, WANG ZhiHua, DU YanMin
    Scientia Agricultura Sinica. 2019, 52(12):  2151-2160.  doi:10.3864/j.issn.0578-1752.2019.12.012
    Abstract ( 291 )   HTML ( 10 )   PDF (379KB) ( 287 )   Save
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    【Objective】 The aim of this study was to establish a scientific evaluation system capable of assessing frozen pear quality. Eighteen parameters of frozen pear quality were determined. 【Method】 Evaluation indices of frozen pear quality were screened by principal component analysis and graded by frequency distribution, and their weight and scoring standard were decided by using an analytic hierarchy process. Discrimination functions were established for frozen pear quality by K-means cluster and discriminant analysis. 【Result】 The results showed that parameters of frozen pear quality dispersed with the coefficient of variation from 9.97% to 360.37%. From the 19 parameters, 7 parameters were chosen to evaluate frozen pear quality, including total soluble solid (TSS), titratable acidity (TA), TSS/TA, peel L *, peel C *, flesh h and stone cell content. The grading standard and the scoring standard of the 7 parameters were established, and five discrimination functions were developed to determine frozen pear quality, which had satisfactory recognition accuracy up to 97.37% (modeling samples) and 100% (test samples). The discrimination functions were very effective in discriminating frozen pear quality. 【Conclusion】The quality of different frozen pear varieties could be comprehensively evaluated using 7 parameters: TSS, TA, TSS/TA, peel L *, peel C *, flesh h and stone cell content. The results of this study indicated that Hongnanguo, Jianbali, Nanguoli, Ranerli, Shatangli, Qiubaili and Huangmacha were the most suitable cultivars to undergo frozen processing.

    Joint Genomic Selection of Yorkshire in Beijing
    ZHANG JinXin, TANG ShaoQing, SONG HaiLiang, GAO Hong, JIANG Yao, JIANG YiFan, MI ShiRong, MENG QingLi, YU Fan, XIAO Wei, YUN Peng, ZHANG Qing, DING XiangDong
    Scientia Agricultura Sinica. 2019, 52(12):  2161-2170.  doi:10.3864/j.issn.0578-1752.2019.12.013
    Abstract ( 363 )   HTML ( 13 )   PDF (1051KB) ( 327 )   Save
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    【Objective】In this study, the molecular breeding via genomic selection was carried out in the joint genomic evaluation on Yorkshire population in Beijing, predicting the breeding value of the new born boars and making selection, so as to improve the selection accuracy of breeding. 【Method】 An admixed population consisting of 4020 individuals from three Yorkshire breeding farms with different genetic background in Beijing was established as the reference group, and the reference animals were selected according to the performance testing records between 2007-2017 in those three pig farms. Three economic traits age at 100 kg (AGE), backfat thickness at 100 kg (BF) and total number born (TNB) were taken into account. The reference and candidate animals were genotyped with Illumina Porcine80K SNP chip. GEBV was estimated by single-step GBLUP (SSGBLUP) method which could make use of both pedigree information and genomic information. GEBVs of candidate boars on the growth traits and reproductive traits were predicted before castration and after performance testing, respectively. Afterwards, the elite candidates were selected according to their GEBVs. Meanwhile, the genetic connectedness among three pig farms was measured by connectedness rating.【Result】Our results showed that the genetic connectedness based on pedigree information among three Yorkshire breeding farms was too low to carry out traditional joint genetic evaluation. However,the genomic relationship coefficients of individuals between farms in G-matrix indicated that genetic links existed among different farms. The genomic selection could realize the joint genomic evaluation through establishing the genetic connectedness via genome-wide markers. A total of 1789 boars were genomic predicted.The accuracy of genomic prediction was largely improved, compared to traditional breeding methods. At the first time of implementing genomic selection or early selection (before the castration of boars), the accuracies of Pedigree Index (PI) for three traits, age at 100 kg (AGE), backfat thickness at 100 kg (BF) and total number born (TNB) were 0.55, 0.56 and 0.41, respectively. However, the accuracies of GEBV from genomic selection were increased to 0.65, 0.70 and 0.60 with improvement of 10, 14 and 19 percentage compared to PI selection, respectively. At the second time of implementing genomic selection (after performance testing), the accuracies of GEBV for AGE, BF and TNB were further increased to 0.78, 0.84 and 0.60, respectively, yielding 8, 12 and 19 percentage higher accuracy than EBV, respectively, in which the accuracies were 0.70, 0.72 and 0.41, respectively. The largest gain of genomic selection was on trait of TNB with low heritability. The early selection based on genomic selection had the same accuracy as traditional selection based on estimated breeding values calculated from performance testing, implying genomic selection could save breeding time and cost with keeping the same accuracy. The comparison of two implementations of genomic selection on 338 boars at different stage showed that the second genomic prediction after performance testing yielded higher accuracy, because the phenotypic records of these boars were also utilized. The accuracies of GEBV for AGE and BF were improved from 0.55, 0.62 to 0.72, 0.84 by increasing 17 and 22 percentage point, respectively. The unbiasedness coefficient was between 0.82 and 1.00, and the unbiasedness of GEBV on traits of AGE and BF were increased from 0.82 and 0.96 to 0.91 and 1.00, respectively. The lower unbiasedness of second genomic selection indicated that the reliability of selecting elite boars was higher.【Conclusion】 Genomic selection could establish genetic connectedness between different farms, enabling joint genetic evaluation which was not feasible in traditional breeding plausible and more breeding farms involved. Compared to traditional PI or EBV selection, genomic selection generated much higher accuracy, and the greatest improvement was obtained on the traits with low heritability. Genomic selection was useful to achieve early selection and to improve the breeding efficiency.

    Transcriptome Analysis of Differentially Gene Expression Associated with Ovary Tissue During the Follicular Stage in Fuqing Goat and Nubian Black Goat
    LI WenYang,LIU Yuan,WU XianFeng,GAO ChengFang,HUANG QinLou
    Scientia Agricultura Sinica. 2019, 52(12):  2171-2182.  doi:10.3864/j.issn.0578-1752.2019.12.014
    Abstract ( 388 )   HTML ( 18 )   PDF (2572KB) ( 199 )   Save
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    【Objective】The aim of this study was to analyze the differentially expressed genes of ovary tissue during follicular stage in Fuqing goat and Nubian black goat.【Method】Transcriptome sequencing of ovary tissue during the follicular stage in Fuqing goat and Nubian black goat were performed by using the Illumina HiSeq TM 2500 platform with 3 biological replicates per goat breed, and verified by quantitative real-time PCR (qRT-PCR). Differentially expressed genes (DEGs) were selected and enriched based on GO, COG and KEGG database. 【Result】A total of 46.68 Gb clean data were obtained in six samples. 149 DEGs were found between Fuqing goat and Nubian black goat, including 53 DEGs up-regulated genes and 96 DEGs down-regulated genes. According to published papers, the genes, including OVN, STAR and EGR1, were considered as candidate genes of fecundity in goats. 108, 30 and 91 DEGs were enriched by GO, COG and KEGG database, respectively. KEGG pathway analysis showed that DEGs annotated to 21 metabolic pathways, and 6 pathways were enriched significantly, such as autoimmune thyroid disease, and allograft rejection, and phagosome and viral myocarditis associated with reproduction traits in goat. 1506 new genes or transcripts were found by BLAST, including 25 DEGs between two breeds. Verified by qRT-PCR, the pattern of selected genes was consistent with the results of transcriptome sequencing, which showed the sequencing results were reliable. 【Conclusion】149 DEGs and 1506 new genes of ovary tissue during follicular stage in Fuqing goat and Nubian black goat were screened by transcriptonal analysis. DEGs between Fuqing goat and Nubian black goat, including OVN, STAR and EGR1, maybe play an important role in goat reproduction traits.

    Expression of p66Shc and Its Relationship with Cytoplasmic Redox Homeostasis in Sheep Oocytes
    ZHANG Tong,LI RuiLan,FAN XiaoMei,LIU ChunJie,HAI RiHan,HUO Min,ZHANG JiaXin
    Scientia Agricultura Sinica. 2019, 52(12):  2183-2192.  doi:10.3864/j.issn.0578-1752.2019.12.015
    Abstract ( 329 )   HTML ( 28 )   PDF (1364KB) ( 172 )   Save
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    【Objective】The aim of this study was to investigate the expression characteristics of p66Shc and its relationship with cytoplasm redox homeostasis in different quality sheep oocytes before and after maturation, which could provide a theoretical basis for revealing the molecular mechanism of p66Shc involved in the regulation of cytoplasmic redox in oocytes.【Method】The sheep cumulus-oocyte complexes (COCs) of ovaries used in this experiment were derived from the slaughterhouse. High quality and poor quality immature oocytes, conventional 24 h matured oocytes and aged oocytes (matured 30 h) were collected, respectively. The expression of p66Shc in different quality sheep oocytes before and after maturation was analyzed by quantitative real-time PCR. The p66Shc protein and mitochondria were co-located by using cellular immunofluorescence combined with MitoTracker Red probe. Simultaneously, the ROS level and the redox homeostasis of different quality oocytes were detected by fluorescence probe DCFH-DA and oocyte spontaneous fluorescence, respectively. In addition, exogenous H2O2-induced oxidative stress was used to treat high quality immature oocytes, and the expression and localization of p66Shc protein were detected and analyzed.【Result】The real-time fluorescent quantitative results showed that the expression of p66Shc mRNA in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes (P<0.05). However, there was no significant difference in the expression of p66Shc mRNA in high-quality oocytes before and after maturation (P>0.05). The co-localization results showed that the regional distribution of p66 protein was consistent with active mitochondrial distribution. The cellular immunofluorescence results showed that the expression of p66Shc protein in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes (P<0.05). Compared with high quality immature oocytes and conventional 24 h matured oocytes, poor quality immature oocytes and aged oocytes showed disordered mitochondrial distribution, decreased activity, increased ROS levels, and imbalance redox homeostasis. In addition, compared with the control group without added exogenous H2O2, exogenous H2O2-induced oxidative stress by treating high-quality immature oocytes significantly (P<0.05) upregulated the expression of p66Shc protein and induced p66Shc from the cytoplasm to the nucleus. 【Conclusion】 The results indicated that the p66Shc gene exhibited high levels of expression in poor quality immature oocytes and aged oocyte, while H2O2-induced oxidative stress significantly up-regulated the expression of p66Shc protein and affected its subcellular localization. In conclusion, the elevated expression of p66Shc perturbed cytoplasmic redox homeostasis in sheep oocytes.