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Table of Content

    16 December 2017, Volume 50 Issue 24
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Genome-Wide Association Analysis of Maize Stalk Anti-Thrust
    MA QingMei, PEI YuHe, CHEN DongBin, SONG XiYun
    Scientia Agricultura Sinica. 2017, 50(24):  4671-4678.  doi:10.3864/j.issn.0578-1752.2017.24.001
    Abstract ( 270 )   HTML ( 8 )   PDF (1450KB) ( 511 )   Save
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    【Objective】SNP loci related to stalk anti-thrust were detected by association analysis between genotype and phenotype data, so that various candidate genes related to stalk anti-thrust were explored.【Method】At present, genome-wide association analysis is widely used in the study of various traits in plants. In this study, 292 maize inbred lines were used as experimental materials, the determination of stalk anti-thrust was carried out in Heze, Zaozhuang, Qingzhou, Jiaozhou in 2015 and 2016. Besides, the plant height, ear height, tassel length and male spike length were measured. On the other hand, the DNA samples of the materials were extracted and then sent to Pioneer seed Products Ltd for genotyping by using MaizeSNP50 gene chip. The chip includes 55126 single nucleotide polymorphism (SNP) markers, after removing the markers whose heterozygous ratio was greater than 10%, loss rate was more than 20% and minimum allele frequency was less than 0.05, there were 25331 SNPs left. Combined with the use of farmCPU, genome-wide association analysis was carried out for analyzing stalk anti-thrust phenotype data, the SNP loci which were significantly related to thrust resistance of stem were detected. According to the physical location of the SNP, the region of the marker was located and then the corresponding candidate genes were obtained. Using SPSS statistics 20 data analysis software, the correlation analysis of plant height, ear height, tassel length and male spike length was carried out to determine the correlation between stem thrust resistance and the 4 traits. In addition, stalk anti-thrust was found to have a significant or extremely significant correlation with plant height, ear height, tassel length and male spike length, but the correlation in each environment was instable, which means that the environment might have a certain impact on the correlation.【Result】A total of 16 SNPs were found to be significantly correlated with stalk anti-thrust, which were located on chromosome box 3.06, 5.02, 4.08, 6.04 and 9.03. SNP loci detected in multiple environments were found to be located on the same chromosome box, for example, SNP loci PZE_108026930, SYN19532, PZE_108048987 and PZE_108050769 which were all found to be located on chromosome 8.03 were detected in Zaozhuang in 2015 and 2016. Moreover, PZE_103111295 and PZE_103125327 found to be located on chromosome 3.06 were detected in Qingzhou in 2015 and Zaozhuang in 2016. A total of 5 candidate genes GRMZM2G504401, GRMZM2G062974, GRMZM2G053767, GRMZM2G348551 and GRMZM2G024260 which encode basic endochitinase A, bZIP transcription factor superfamily protein, 40S ribosomal protein S4-like protein isoform X1, soluble starch synthase 2-3, adenine nucleotide alpha hydrolase-like superfamily protein were also obtained, respectively. 【Conclusion】GRMZM2G062974 is involved in the expression regulation of plants under stress and responds to a variety of abiotic stresses. GRMZM2G348551 participates in the pathway of glucose metabolism and regulates the synthesis of amylopectin, which is involved in the formation of cell walls. GRMZM2G504401 is induced by biotic or abiotic stresses in plant. Lodging is affected by its own or a variety of stresses, and the functions of these genes are compatible with lodging resistance, so future studies should focus on the function of these genes.
    Comprehensive Evaluation of Germplasm Resources of Upland Cotton in Xinjiang
    LIU XiangYu, ZHAO Long, BAHARGUL·Xamxi, PENG Hua, ABDUREYIM·Ibrayim
    Scientia Agricultura Sinica. 2017, 50(24):  4679-4691.  doi:10.3864/j.issn.0578-1752.2017.24.002
    Abstract ( 333 )   HTML ( 3 )   PDF (779KB) ( 273 )   Save
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    【Objective】Based on the morphological index, yield index and quality index, this study comprehensive evaluated the performance of the cotton germplasm resources in Xinjiang under the condition of seasonal water deficit in order to determinate main upland cotton cultivars and lay the foundation for the cotton cultivars improvement in Northwestern arid region.【Method】The comprehensive evaluation of quantitative traits about plant height, number of fruit branch, breeding time, effective boll number per plant, boll weight, lint percentage, seed index, seed cotton yield, lint yield, fiber length, fiber uniformity, fiber strength, fiber elongation, micronaire, fiber reflectance, yellow degree and SCI of 126 cotton varieties under the condition of seasonal water deficit were evaluated by correlation analysis, principal components analysis, hierarchical cluster analysis, stepwise discriminant analysis and multivariate analysis of variance etc.【Result】Firstly, the results of the correlation analysis showed that there is a certain correlation and overlap of information between 17 quantitative traits, because 56 of these coefficients is extremely significantly correlation at P<0.01 and 22 of these coefficients is significantly correlation at P<0.05. Secondly, the results of the principal components analysis showed that the first eight principal components represent 86.34% of the information which is the 17 quantitative traits of the 126 cotton varieties. The contribution rate is respectively 27.45%, 17.18%, 11.61%, 8.42%, 6.66%, 5.33%, 5.08%, and 4.63%.Thirdly, the results of the hierarchical cluster analysis showed that the 126 cotton cultivars were clustered into seven categories when the class separation distance is 12.5. Among them, the first classⅠ has 22 varieties, the class Ⅱ has 17 varieties, the class Ⅲ has 19 varieties, the class Ⅳ has 28 varieties, the class has 19Ⅴ varieties, the class Ⅵ has 13 varieties, the class Ⅶ has 8 varieties. Fourthly the results of the stepwise discriminant analysis showed that the results of cluster analysis are accurate and reliable, because 114 cotton varieties were correctly discriminated, which identification rate was 90.48% and 12 cotton varieties were incorrectly discriminated, which false identification rate was 9.52%. Finally, the results of the multivariate analysis showed that the seven categories of cotton varieties were evaluated scientifically and the corresponding improvement schemes were proposed, which were the classⅠwhich is the cultivars of middle yield and middle quality, the classⅡwhich is the cultivars of low yield and high quality, the class Ⅲ which is the cultivars of high yield and high quality, the classⅣwhich is the cultivars of middle- and high- yield and middle quality, the class Ⅴ which is the cultivars of middle- and high- yield and upper middle quality, the class Ⅵ which is the cultivars of high yield and middle quality and the classⅦ which is the cultivars of low yield and low quality.【Conclusion】On the one hand, the work of the cotton breeding in production in Xinjiang has a lot to improvement; On the other hand, most of the cultivars fall short of the requirements of high count cotton, besides the class Ⅵ and the class Ⅴ. The main upland cotton cultivars which are Suitable to Spin middle Count Cotton can be selected in the classⅢ. The classⅡare suitable to spin the high-quality cotton of high tenacity, but their yield indicators need to be improved. In future, in order to achieve the optimality which include yield and quality, the work of the cotton breeding in Northwestern arid region should be done by selecting suitable breeding strategies, according to different category of varieties.
    Construction of Cotton Variety Fingerprints Using CottonSNP63K Array
    SUN ZhengWen, KUANG Meng, MA ZhiYing, WANG XingFen
    Scientia Agricultura Sinica. 2017, 50(24):  4692-4704.  doi:10.3864/j.issn.0578-1752.2017.24.003
    Abstract ( 251 )   HTML ( 28 )   PDF (10077KB) ( 163 )   Save
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    【Objective】 The objective of this study is to screen genomic-specific SNPs, considering the SNP with single loci and the reference genomic sequence information of upland cotton TM-1. 【Method】 Based on 719 natural germplasm resources with plentiful genetic backgrounds, using the CottonSNP63K array developed by Illumina, quality control of the original genotyping data obtained from chip scanning was performed by GenomeStudio software, the genotyping data of the SNP locus of the tested samples were obtained. According to the two published Gossypium hirsutum TM-1 genome versions, including the G. hirsutum (AD1) genome BGI v1.0 of Cotton Research Institute of Chinese Academy of Agricultural Sciences and the G. hirsutum (AD1) genome NBI v1.1 of Nanjing Agricultural University, the genome-wide BLAST analysis was performed on the flanking sequences of each SNP with CottonSNP63K (63 058 SNPs) array to screen specific SNP loci with single copy, and these SNPs were used for the construction of fingerprints. 【Result】 SNP genotyping of 719 materials using CottonSNP63K was classified into SNP locus without detectable signal, SNP locus without polymorphism and the polymorphism SNP loci. The polymorphism SNP loci could be divided into double-site SNP, multiple-site SNP and single-site SNP (genomic-specific SNP). The results of the Blast analysis with the two upland cotton TM-1 reference genome sequences showed that there were 5 474 specific SNP markers in the BGI v1.0 version, while NBI v1.1 TM-1 version only 1 850, and the common specific SNPs of both were 1 594. Further considering the three evaluation indexes including the genotyping effect, call rate and polymorphism, the SNPs with scores≥0.7, call frequency≥0.95 and MAF≥0.2 were screened, and 471 specific SNPs with high call rate and high polymorphism were obtained. Among these SNP loci, 430 were on chromosomes and 41 were on scaffolds. Taking into account of the degree of linkage between the markers, finally 393 core SNP loci were obtained after removing 37 linkage markers, and the DNA fingerprints of 719 resource materials were constructed by using 393 core SNPs. The results showed that 97% of the materials could be accurately and effectively identified except for some accessions with similar genetic background. 【Conclusion】 A total of 393 genomic-specific SNPs were screened out, and these core SNPs were used to construct the DNA fingerprints of 719 resource materials. This study will provide a reference for application of SNP molecular markers in genetic improvement of important cotton traits.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Re-Evaluation of Using the Color Difference Between the Top 3rd Leaf and the 4th Leaf as a Unified Indicator for High-Yielding Rice
    LING QiHong, WANG ShaoHua, DING YanFeng, LI GangHua
    Scientia Agricultura Sinica. 2017, 50(24):  4705-4713.  doi:10.3864/j.issn.0578-1752.2017.24.004
    Abstract ( 234 )   HTML ( 2 )   PDF (345KB) ( 582 )   Save
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    【Objective】 To re-evaluate if the value of color different between the top 3rd leaf and the 4th leaf would be a unified indicator for high-yielding rice.【Method】Two experiments were carried out with different leaf color cultivars, including four japonica rice cultivars (Jinnanfeng, 9915, Yueguang, 9325) and two indica rice cultivars (Baidao, H97-32) in 2001 and 2002. One experiment applied different base nitrogen fertilizer as 0, 6.5, 13.0, 19.5 and 26.0 g·m-2. Another experiment applied different panicle nitrogen fertilizer as 0, 6.5, 13.0, 19.5 and 26.0 g·m-2, followed by base nitrogen as 13.0 g·m-2. SPAD values of top 4 leaves were measured using the Chlorophyll meter SPAD-502 at N-n stage, the 2nd leaf-age from top and full heading stage. The relationship of the different SPAD value of the top 4th leaf to the top 3rd leaf with plant nitrogen content and yield was investigated. 【Result】 It is difficult to diagnose rice nitrogen status base on single leaf’s SPAD value, because the value is changing if cultivar or rice growth period changed. The three kinds of leaf color differences of top in each growth stage showed: top 4 top 3, which were the exact physiological reflection of nitrogen deficiency, normal and excess. There is no regular relationship of the SPAD of single leaf with tillering rate, the percent of tillers to panicles, spikelet number per panicle and setting rate, so the SPAD of single leaf can’t be used as a common diagnostic indicator. However, there is a regular relationship between different value form the top 3rd leaf to the 4th leaf and yield components. Furthermore, rice should have highest yield if the different value is zero at N-n stage, the 2nd leaf-age from top and full heading stage. 【Conclusion】 The difference color between the top 3rd leaf and the 4th leaf is the internal indicator of nitrogen nutrition level in rice. It can diagnosis rice nitrogen nutrition situation well and truly with comparing color of the top 3rd and 4th leaf, coordinating diagnosis single leaf’s color.
    Construction and Application Effect of the Leaf Value Model Based on SPAD Value in Rice
    LI Jie, FENG YueHua, MOU GuiTing, XU GuiLing, LUO QiangXin, LUO KangJie, HUANG ShiFeng, SHI Xin, GUAN ZhengCe, YE Yong, HUANG YouGang
    Scientia Agricultura Sinica. 2017, 50(24):  4714-4724.  doi:10.3864/j.issn.0578-1752.2017.24.005
    Abstract ( 288 )   HTML ( 3 )   PDF (386KB) ( 348 )   Save
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    【Objective】The relationship of different plots, nitrogen application amount, SPAD value derivative index and yield was studied to construct linear model of nitrogen rate based on SPAD value in rice, so as to easily, quickly, perfectly recommend nitrogen rate.【Method】The experiment was conducted by using Qyou6 as the materials with four nitrogen levels (0, 75, 150, 225 kg?hm-2) in 2015 and 2016. We compared the relationship between yield and apparent nitrogen supply of field, and between SPAD value derivative index and apparent nitrogen supply of field in this experiment. And the effect of variable nitrogen application based on leaf value model was also studied.【Result】The results showed that, curve relationship between yield and apparent nitrogen supply of field at heading stage was extremely significant positive correlation, and R2 of two years was 0.5523, 0.7148, respectively. In this fitting relationship, the higher yield of every years was 9 264.93 kg?hm-2, 11 167.97 kg?hm-2. The difference of maximum yield was 1 903.14 kg?hm-2 in two years, and yield in 2016 was 20.54% higher than that in 2015. But the apparent total nitrogen uptake of the highest yield in different years were close, which was 575.27 kg?hm-2, 546.71 kg?hm-2, respectively. And the difference of apparent total nitrogen uptake was only 28.56 kg?hm-2, and apparent total nitrogen uptake was -4.96% higher than in 2015. The linear relationships between SPADL3 (SPAD value of third leaf), SPADL4 (SPAD value of fourth leaf), SPADmean (average SPAD value of four leaves), SPADL3×L4/mean (product of SPAD value of third leaf and SPAD value of fourth leaf divided by average SPAD value of four leaves)and apparent nitrogen supply of field were significant or extremely significant under different growth period and year. On single leaf, the change of slope of linear relationship between SPADL3 and apparent nitrogen supply was smaller at jointing stage in years, and value of slope was 0.0156 in 2015, 0.0154 in 2016, respectively. At heading stage, the slope of linear relationship between SPADL1 and apparent nitrogen supply in 2015 and 2016 was 0.0172, 0.0173, respectively. At joining and heading stage of different years, the slope and origin of linear relationship between SPADL3×L4/meanand apparent nitrogen supply of 2016 increased by  -28.70%, 17.41%; -15.34%, 56.11% than that of 2015, respectively. Constructing a new rice nitrogen model based on SPAD value, which was called the leaf value model. The total nitrogen rate of leaf value model was difference between apparent total nitrogen uptake and apparent nitrogen supply of soil, and it could estimate apparent nitrogen supply of soil by measured value of SPAD value derivative index. And R2 of linear fitting between SPAD value derivative index and apparent nitrogen supply of field at heading stage was higher than that at jointing stage. At joining stage, the total nitrogen rate which was estimated by SPADL4 or SPADL3×L4/mean was close with that was estimated by SPADL3 or SPADmean, and the former was 50% higher than the later. The effect of variable nitrogen application based on leaf value model showed that yield of variable area was 820.68 kg?hm-2 higher than that of control area, and nitrogen partial productivity and nitrogen agronomic efficiency of variable area was 13.74%, 103.45% higher than that of control area, respectively. A general expression for leaf value model was Nw=Nz-[(Ys-b)/k-Ng], where Nw, Nz, Ys, Ng meant total nitrogen application rate (kg?hm-2), apparent total nitrogen uptake (kg?hm-2), SPAD value derivative index, base-tiller fertilizer amount (kg?hm-2), respectively, and k, b was slope and origin of linear relationship betweent apparent nitrogen supply of field (Nx) and SPAD value derivative index (Ys), and Nx was sum of nitrogen content of soil (kg?hm-2) and already manure amount. 【Conclusion】Variable nitrogen application based on leaf value model could reduce yield difference and increase yield, nitrogen agronomic efficiency, nitrogen partial productivity and nitrogen contribution rate.
    PLANT PROTECTION
    Molecular identification and specific detection of Telosma mosaic virus infecting passion fruit
    XIE LiXue, ZHANG XiaoYan, ZHENG Shan, ZHANG LiJie, LI Tao
    Scientia Agricultura Sinica. 2017, 50(24):  4725-4734.  doi:10.3864/j.issn.0578-1752.2017.24.006
    Abstract ( 389 )   HTML ( 10 )   PDF (1903KB) ( 635 )   Save
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    【Objective】 The objective of this study is to identify Telosma mosaic virus (TeMV) on passion fruit in Fujian orchard, develop a molecular method for specific and rapid identification of TeMV, and to provide a reference for prevention and control of the virus.【Method】The Fujian passion fruit samples were detected by using the serology method, electron microscopic observation, universal degenerate primers RT-PCR and specific primers RT-PCR. The PCR products of the positive sample were cloned and sequenced. A set of specific primes amplified the total length of coat protein (CP) gene was designed according to the sequences of reported TeMV and sequence determination of this study, and then specific primers RT-PCR detection method was established after optimizing reaction conditions. The sequence determination results were analyzed with BLAST program and DNAMAN software, and the phylogenetic tree was constructed based on the CP gene sequences obtained in this study by using Bayesian inference (BI) method implemented in MrBayes. 【Result】The serology results showed that one passion fruit sample exhibiting mosaic and crinkle symptom reacted with Potyvirus antiserum. The positive sample was found to have about 750 nm×12 nm linear virions by electron microscopic observation. The expected fragment was amplified from the positive sample by universal degenerate primers RT-PCR, and then cloned and sequenced. Sequence analysis showed that the obtained sequence from the positive sample was identical with the expected size (680 bp), and shared the highest nucleotide sequence identity (98.2%) with the reported TeMV gene sequence. The full-length sequence of CP gene obtained by specific primers RT-PCR was 816 bp (named BXGFJ-13 isolate), and the sequence of nucleotide and amino acid of BXGFJ-13 was 86.2%-98.4% and 88.2%-97.8% identity, respectively, with the reported TeMV isolates. The results of phylogenetic analysis showed that the 13 TeMV isolates could be divided into 3 groups, and the same area or host derived isolates preferentially clustered together, suggesting these isolates had a strong geographical and host specificity. BXGFJ-13 isolate obtained in this paper and Guangxi isolate of China (KJ789129) clustered into a branch with high posterior probability, and then clustered together with two Thailand isolates (AM409188, AM409187) into the 2nd group (Group II), showing that BXGFJ-13 and Guangxi isolate had the closest phylogenetic relationship. The specific primers RT-PCR showed good specificity, which only amplify the expected fragment from TeMV-infected passion fruit sample, and no expected fragment was obtained from Cucumber mosaic virus (CMV), Beet mosaic virus (BtMV), Soybean mosaic virus (SMV), Ornithogalum mosaic virus (OrMV), Onion yellow dwarf virus (OYDV), East Asian Passiflora virus (EAPV) and healthy control. The sensitivity results showed that the target fragment could be amplified from diluted 102 fold RNA.【Conclusion】According to the species demarcation criteria for the Potyvirus given by International Committee on Taxonomy (ICTV) and the results of serological detection and electron microscopic observation, TeMV on the passion fruit sample exhibiting mosaic and crinkle symptom in Fujian orchard was confirmed. The establishedassay of specific RT-PCR could be used for rapid detection of TeMV.
    Effects of Imidacloprid Flowable Concentrate for Maize Seed Coating on the Arthropod Community and the Main Non-Targeted Insect Pests in Maize Field
    LI GuanNan, MIAO ChangJian, LI WeiZheng, WANG GaoPing, LIU XiaoLan, GUO XianRu, YAN FengMing
    Scientia Agricultura Sinica. 2017, 50(24):  4735-4746.  doi:10.3864/j.issn.0578-1752.2017.24.007
    Abstract ( 212 )   HTML ( 5 )   PDF (780KB) ( 468 )   Save
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    【Objective】The objective of this study is to investigate the effects of imidacloprid flowable concentrate for seed coating (FSC) on the arthropod community and the non-targeted insects in maize field, in order to provide a theoretical basis for the application of seed coating agent.【Method】The study was conducted in the experimental field in Xuchang Campus of Henan Agricultural University from June to September in 2015 and 2016, respectively. Maize seeds were coated with the recommended dose of 600 g·L-1 imidacloprid FSC and its doubled and quadrupled dose, with water as control. Each treatment was repeated for 4 times and arranged with randomized block. The density of the arthropods in maize field was estimated by using 5 sampling spots methods, each spot with 10 tagged plants at seedling, bellmouthed, booting and filling stages of the maize plants. The characteristics of the arthropod community including arthropod species and their amounts in different treatment areas were analyzed. In order to analyze the effects of imidacloprid FSC on non-targeted insects, the amount of insect pests in maize ears within the sampling spots at filling stage and the amount of lepidopteran larvae in maize ears and stalks by sampling 100 plants within two lines at mature period were surveyed. 【Result】Imidacloprid FSC treatment had no effect on the number of species of arthropod in maize field, and the species richness in the treatment areas showed similar change trend to that in the control area. The difference of the richness in different treated plots at the same time was related to the low density and uneven distribution of some species. Seed coating could not lead to the decrease of the diversity and evenness of the arthropod community. When the population of target pest, Rhopalosiphum maidis, occurred at high level, the diversity and evenness in seed coating areas could be increased by inhibiting the target pests. The effects of imidacloprid FSC on the dominant species differed with maize growth stages in different years. In 2015, R. maidis occurred at higher population density, the dominant species of the phytophagous insects and predators in seed coating plots were similar to that in control plot at early growth stage of maize, while Pseudaletia separata and Erigonidium graminicolum were the dominant species in plots of 2 times and 4 times doses of maximum recommended doses (MRD). In 2016, when R. maidis occurred at lower population density, Ostrinia furnacalis and Helicoverpa armigera were dominant phytophagous species, and Propylea japonica and Harmonia axyridis became the dominant species of predators. The dominance degree showed similar change trend to dominant concentration index. The impact of seed coating on ear-feeding lepidopteran larvae was complex. For example, the population density of O. furnacalis in the treated plots was higher than or equivalent to that in the control plots during early and middle grain-filling, and the density of Dichocrocis punctiferalis larvae also increased significantly at the early grain-filling stage, but decreased at the middle grain-filling stage. For P. separata, the density in seed coating plots increased significantly than that in control plot at middle filling stage, while the density of H. armigera in seed coating plots differed from the check plots with no remarkable degree. The results of the two-year investigations showed that the density of O. furnacalis, D. punctiferalis and P. separata had an increasing trend in seed coating plots, but it was not closely related to the dosage of seed coating agent of 600 g·L-1 imidacloprid. 【Conclusion】It is recommended that lepidopteran pests should be prevented or controlled in the maize field with application of imidacloprid coating agent.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    The Effects of Soil Water Content on N2O Emissions and Isotopic Signature of Nitrification and Denitrification
    ZHENG Qian, DING JunJun, LI YuZhong, LIN Wei1, XU ChunYing, LI QiaoZhen, MAO LiLi
    Scientia Agricultura Sinica. 2017, 50(24):  4747-4758.  doi:10.3864/j.issn.0578-1752.2017.24.008
    Abstract ( 283 )   HTML ( 8 )   PDF (486KB) ( 694 )   Save
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    【Objective】 The objective of this paper is to understand the contribution of microbial processes to N2O production and its changing rules under different water contents to provide a theoretical basis for reducing agricultural N2O emissions. 【Method】A microcosm experiment was performed to investigate the effects of different water-filled pore space on N2O emissions and isotopic signatures (δ15Nbulk, δ18O and nitrogen isotopomer site preference of N2O) of soil at Shunyi district, Beijing. The study combined stable isotope technique and gas inhibitor method to analyze N2O flux and its isotope signatures that emitted from soil. The experiment was set up three different water content levels, including 67%, 80% and 95% WFPS, and with three inhibitor levels, (without C2H2, with 0.1% (V/V) C2H2 and with 10% (V/V) C2H2). After two hours incubation, the soil samples were collected to determine the concentrations of NH+ 4-N and NO- 3-N. The gas samples were collected to determine the isotope signatures, and the two end-members mixing model was applied to quantify the respective contributions of microbial processes to N2O production. 【Result】According to the incubation of the soil, the weighted average N2O flux of 95%, 80% and 67% WFPS were 1.17, 0.27 and 0.08 mgN·kg-1·d-1, respectively, and the N2O emissions of 95% WFPS were significantly higher than that of both 80% and 67% WFPS, as well as the N2O emissions of 80% WFPS were significantly higher than that of 67% WFPS. The cumulative emissions of (N2O+N2) in 95%, 80% and 67% WFPS were 18.05%, 5.27%, and 1.24% of initial mineral nitrogen, respectively, over the entire incubation period. The cumulative emissions of (N2O+N2) were 19.61, 5.72 and 1.35 mgN·kg-1, respectively; the initial content of NH+ 4-N+NO- 3-N was 108.62 mgN·kg-1. Compared with 67% WFPS, the cumulative (N2O+N2) emissions of 95% and 80% WFPS increased 13.53 and 3.24 times, respectively. The cumulative emissions of (N2O+N2) in 95% WFPS was 2.43 times greater than that of 80% WFPS. The values of reduced NH+ 4-N+NO- 3-N as gaseous nitrogen increased with the increase of the water content. The weighted average δ15Nbulk values varied from -42.93‰ to -4.07‰, and the higher level of soil water content showed significantly higher N2O emissions. 10% (V/V) C2H2 would inhibit the reduction of N2O to N2. The δ18O values with 10% (V/V) C2H2 were significantly smaller than that of with 0.1% (V/V) C2H2 in three water content levels. And the ratio of N2O/(N2O+N2) reduced with the increase of soil moisture. Multiple N2O processes occurred simultaneously in all treatments. The values of SP increased during the initial four days and then decreased gradually with incubation time. The SP values of 67%WFPS treatment at the first two days ranged from 6.74‰ to 12.04‰, and the contribution of denitrification to N2O production was from 56.36% to 66.15%, suggesting that denitrification was the dominant microbial process, then the contribution of nitrification (55.78%-100%) to N2O production became greater. The weighted average SP value was 10.26‰ in 80% WFPS treatment, indicating denitrification (40.90%-74.04%) was the major N2O production process. There were larger SP values in 95% WFPS treatment with 10% (V/V) C2H2 in the first seven incubation days, ranged from 7.61‰ to 21.11‰. Compared with 0.1% (V/V) C2H2, the weighted average SP values of N2O under 95%, 80% and 67% WFPS treatments with 10% (V/V) C2H2 produced from soil reduced by 0.10, 0.33 and 0.06 times respectively.【Conclusion】 The increase of soil water content promotes N2O emission, and the 95% WFPS treatment showed the highest N2O emissions. In the 67% WFPS treatment, the initial stage of N2O emission was dominated by denitrification, followed by nitrification. Denitrification was the dominate process in 80% WFPS treatment and nitrification was the dominate process in 95% WFPS treatment.
    Response of Dry Land Soil Respiration to Conservation Tillage Practices During Drying-Wetting Cycles
    DING XinYu, WANG ZiKui, YANG Xuan, DU ShanShan, SHEN YuYing
    Scientia Agricultura Sinica. 2017, 50(24):  4759-4768.  doi:10.3864/j.issn.0578-1752.2017.24.009
    Abstract ( 226 )   HTML ( 2 )   PDF (451KB) ( 474 )   Save
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    【Objective】This study was conducted to investigate the effects of conservation tillage practices on soil respiration during drying and wetting period in dry farmland. 【Method】A long-term conservation tillage experiment was carried out at the Qingyang Experimental Station of Lanzhou University. The experiment had four treatments: conventional tillage (T), conventional tillage with stubble retention (TS), no-till without stubble retention (NT) and no-till (NTS). Soil respiration rate was measured continuously with LI-8100 automated soil respiration system during the common vetch growing season from July 7 to August 20 in the 2014. 【Result】The mean soil respiration rates of T, TS, NT and NTS during the drying period were 2.16, 3.56, 2.26 and 2.45 µmol·m-2·s-1, respectively, and the corresponding values during the wetting period were 2.09, 5.31, 2.80 and 3.56 µmol·m-2·s-1, respectively. Diurnal patterns of soil respiration after the heavy rainfall event were different among the four tillage practices. Soil respiration was positively related to soil water content and negatively related to soil temperature during the drying period, but had an opposite relationship with these parameters after the rewetting. The sensitivity of soil respiration to soil temperature (Q10) reduced by no-till and stubble retention practices. During the wetting period, the Q10 values of T, TS, NT and NTS were 1.37, 1.24, 1.31 and 1.25, respectively, and dropped to less than 1.0 for all treatments during the dry period. 【Conclusion】No-till has the potential to release less CO2. Residue retention buffers soil temperature and soil water content fluctuations, and reduces the Q10 value. Our results also illustrated that it is fairly important to consider the short-term changes of soil respiration during the drying-wetting cycles when modeling the relationship between soil respiration and its influencing factors.
    HORTICULTURE
    Effects of Fertilizer Being Bag-Controlled Released on Root Growth, Nitrogen Absorption and Utilization, Fruit Yield and Quality of Peach Trees
    ZHANG YaFei,LUO JingJing, PENG FuTian, GAO HuaiFeng, WANG GuoDong, SUN XiWu
    Scientia Agricultura Sinica. 2017, 50(24):  4769-4778.  doi:10.3864/j.issn.0578-1752.2017.24.010
    Abstract ( 235 )   HTML ( 7 )   PDF (458KB) ( 261 )   Save
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    【Objective】In order to provide reference for scientific fertilizing in peach orchard, the effects of fertilizer being bag-controlled released and fertilizer spread application treatments on dynamic changes of available nutrients in soil, root growth and horizontal distribution, nitrogen utilization rate and fruit yield and qualities were studied.【Method】 Late-ripening peach ‘Ruipan 21’ were used as experimental materials. The field experiment was conducted from 2012 to 2016 with three fertilization modes: bag-controlled release fertilizer (BCRF), equal amounts of fertilizer spreading application in one time (FSA I) and high amounts of fertilizer spreading application in two times (HFSA II). The soil available nutrients contents were determined in April, July and October every year, and the fruit yield and qualities were investigated from 2014 to 2016. The nitrogen absorption and distribution in peach plants were studied by using 15N tracer technique, and the root growth and distribution were investigated in 2016. 【Result】 The soil available nutrients, including available phosphorus, available potassium and available nitrogen of BCRF treatment were steady for five years, however the soil available nutrients of FSA I and HFSA II treatments were higher at the early stage and then showed a trend of decline. The roots of BCRF treatment horizontally distributed in the range of 100 cm from the trunk and that of FSA I and HFSA Ⅱ treatments could grow to the range of 140 cm and 160 cm. Moreover the fine roots of BCRF treatment has a higher proportion, accounting for 83.95% of the total roots’ length compared with the FSA I and HFSAⅡ treatments, accounting for 75.16% and 70.63%. In addition, the BCRF treatment could maintain the stability of the biomass of the root system. The plant nitrogen utilization rate for BCRF treatment was 1.39 times and 1.81 times of FSA I and HFSA Ⅱ treatments, respectively. The yield of BCRF treatment was increased by about 20% of FSA I, and has no significant difference with HFSA Ⅱ treatment, but the fruit quality of BCRF treatment was significantly improved. 【Conclusion】 Under the treatment of fertilizer being bag-controlled released for 5 years, the soil nutrient levels were remained stable, the occurrence of fine roots were promoted, the root longevity was prolonged, and the roots distribution was concentrated to form “Intensive Root System”. Therefore the nitrogen utilization rate of peach plant of BCRF treatment was increased and the fruit yield and qualities were improved.
    Cloning and Functional analysis of lycopene β-cyclase promoter of marigold (Tagetes erecta)
    ZHANG ChunLing, WANG NiHui, WANG NingLe, BAO ManZhu, HE YanHong
    Scientia Agricultura Sinica. 2017, 50(24):  4779-4789.  doi:10.3864/j.issn.0578-1752.2017.24.011
    Abstract ( 268 )   HTML ( 4 )   PDF (5209KB) ( 670 )   Save
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    【Objective】The objective of this study is to clone Lycopene β-cyclase enzyme(LCYB) gene and its promoter from marigold(Tagetes erecta), to analyze their bioinformatics characters and predict the promoter function. This study provides a reference for the regulation of the metabolic mechanism of the carotenoids and the control of the lutein concentration in marigold.【Method】The cDNA sequence of TeLCYb gene was cloned using RT-PCR method. Bioinformatics tools were applied to analyze both the TeLCYb gene sequence and the characteristics of its encoded protein sequence. DNAMAN and online software BoxShade were used to make multiple sequence alignments between the TeLCYb amino acid sequence and their homologous sequences, and MEGA6.0 was used to constructed phylogenetic tree of homologous species. The promoter sequence was cloned according to its cDNA sequence using FPNI-PCR. Thereafter, two different 5′ UTR deletion mutants of the TeLCYb gene promoter were amplified by PCR, and then were inserted into the vector V152 to construct promoter-GUS fusion genes expression vectors, named pTeLCYb(-1969)::GUS and pTeLCYb(-1140)::GUS, respectively, and then were transformed into tobacco by agrobacterium tumefaciens-mediated transformation of tobacco, respectively. And the promoter activities were quantitatively estimated using GUS report gene.【Result】The TeLCYb was successfully cloned from marigold(Tagetes erecta). The bioinformatics analysis showed that its full length was 1 865 bp with an open reading frame (ORF) of 1 527 bp length, encoding 508 amino acids. Homology analysis showed that the deduced TeLCYb protein was highly homologous to other LCYb proteins from Chrysanthemum morifolium,Dendranthema lavandulifoliu and TeLCYb protein has typical protein special elements of LCYb protein. Phylogenetic analysis also indicated that TeLCYb was clustered into the branch with asteracea. A 1 806 bp promoter sequencewas obtained via FPNI-PCR. Bioinformatics analysis of promoter sequence revealed that this fragment contained some promoter core elements, such as TATA-box and CAAT-box, and 13 light responsive elements and 5 hormones responses elements. Meanwhile, MYB transcription factor binding elements and heat-resistant response elements, physiological control elements and cis-acting regulatory elements were found in this sequence. Functional characterization of promoters by constructing pTeLCYb (-1969)::GUSand pTeLCYb (-1140)::GUS plant expression vectors and transforming into tobacco respectively showed that both promoters were active in stems, petals, anthers and capitals. But there were significant differences of GUS enzyme activity among plant tissues, and blue spots found in anthers and capital were significantly stronger than that of petals. Compared with the expression level of pTeLCYb (-1969)::GUS, the shorter sequence of pTeLCYb (-1140) led to a significant increase in the activity of GUS in the transgenic leaves and root and sepals were also found GUS enzyme activity. 【Conclusion】 Different length of promoters can drive the expression of GUS gene, but there are differences in the sites and intensity where promoters functions, we hypothesize that a positive regulatory factor of light response elements maybe exist between 1 140 bp and 164 bp area of 5′ upstream to ATG, while hormone response elements and the heat stress response element may suppress or reduce the promoter function.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Establishment and Application of Loop-Mediated Indirect PCR Assay Based on Single-Strand Substitution for Detection and Differentiation of PEDV and TGEV
    ZHENG Ming, LI HuaWei, LIU YingYing, WANG YongFen, BIAN ChuanZhou, GUO HongWei
    Scientia Agricultura Sinica. 2017, 50(24):  4790-4798.  doi:10.3864/j.issn.0578-1752.2017.24.012
    Abstract ( 221 )   HTML ( 8 )   PDF (936KB) ( 279 )   Save
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    【Objective】 The virus diarrhea of pigs is an acute and highly contagious enteric disease, which is mainly caused by the porcine epidemic diarrhea virus (PEDV) and the porcine transmissible gastroenteritis virus (TGEV). The clinical signs of the disease mainly include vomiting, diarrhea, dehydration, high morbidity, and mortality. PEDV and TGEV belong to two distinct species of the Alphacoronavirus genus within Coronaviridae. Pigs of all ages are susceptible, especially the piglets, which can lead to death of large numbers of piglets and cause huge economic losses to the swine industry. Due to the clinical symptoms, pathologic changes and epidemiology are very similar, it is difficult to distinguish them by clinical diagnosis. Therefore, the rapid, specific preclinical identification of PEDV/TGEV is of great significance for preventing the outbreak and spread of this disease. The aim of this study is to establish the method of loop-mediated indirect PCR assay for the detection of PEDV/TGEV and the etiologic diagnosis for viral diarrheas in piglets. 【Method】According to the genome sequence information of PEDV and TGEV in GenBank database, a highly conserved region of PEDV and TGEV nucleoprotein (N) gene sequences were selected based on the homology comparison of the two viral genes. Two pairs of specific adjacent probes were designed, which were labeled on both ends of TOC1 gene fragments of different sizes as specific probe-labeled reporter genes for loop-mediated indirect PCR. After hybridizing with the target genes and cyclizing, the probe-labeled reporter genes were amplified by reverse PCR. The loop-mediated indirect PCR assay had been developed for the detection of PEDV/TGEV, and the specific PCR products were 404bp for PEDV and 252bp for TGEV, respectively.【Result】 The experimental results showed that the assay could be useful for the specific detection of PEDV and TGEV, from a contaminated sample by any one of them or by both. The specificity and sensitivity of the loop-mediated indirect PCR assay were tested. The results showed that the specificity was high, and no cross-amplification was observed with other common swine-borne viruses such as PCV2, RAV, PRRSV, CSFV, PRV and PPV. The lowest detection limits of the loop-mediated indirect PCR assay were 1.6 pg /μL for PEDV and 8 pg /μL for TGEV, respectively. And simultaneous detection of both pathogens did not affect the detection sensitivity. The comparison test was conducted on 157 clinical samples collected from adjacent pig farms by loop-mediated indirect PCR, conventional PCR and SYBR Green real-time RT-PCR. The results showed that the PEDV positive detection rates of loop-mediated indirect PCR, conventional PCR and SYBR Green real-time RT-PCR were 33.76%, 21.66% and 36.31% respectively, and the TGEV detection the positive rates were 26.75%、13.38% and 28.03% respectively. Results of Kappa analysis showed that, for both PEDV and TGEV detection, overall agreements between the loop-mediated indirect PCR and the SYBR Green real-time RT-PCR were both 96.18% with a kappa value of greater than or equal to 0.90.【Conclusion】The study suggested that the established loop-mediated indirect PCR was a rapid, accurate, sensitive and specific etiologic diagnosis tool, suitable for the differential diagnosis of PEDV and TGEV.