Table of Content

01 May 2018, Volume 51 Issue 9

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Identification and Gene Mapping of a Shorten Panicle and Seed Mutant sps1 in Rice (Oryza sativa L.)

XIE Jia, ZHANG XiaoBo, TAO YiRan, XIONG YuZhen, ZHOU Qian, SUN Ying, YANG ZhengLin, ZHONG BingQiang, SANG XianChun

Scientia Agricultura Sinica. 2018, 51(9):  1617-1626.  doi:10.3864/j.issn.0578-1752.2018.09.001

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【Objective】Identification and gene mapping of a shorten panicle and seed mutant is significant for rice functional genomics research and molecular breeding. 【Method】A shorten panicle and seed mutant (sps1) was identified from the EMS-induced library. At the maturity stage, agronomic traits such as plant height, panicle length, seed number per panicle, filled grain number per panicle, seedsetting rate and 1000-seeds weight were measured. Scanning electron microscopy was carried out to analyze the inner and outer epidermis between the wild type and sps1 and the grain morphology of wild type and sps1 was observed by paraffin section. The sps1 was crossed with Jinhui 10, and the F₁ and F₂ generations were used for genetic analysis. Sensitivity test of brassinolide (BR) was carried out on sheath of wild-type and sps1. The root, stem, leaf, sheath and spike were collected, and the genes associated with grain development and the genes associated with BR were analyzed in qPCR.【Result】The sps1 was dwarf in all phases of plant development, and the panicle length, the first, second and third internodes were significantly shorter than those of the wild type. In addition, there were significant decrease in the branch number, seed-setting rate and the 1000-seeds weight in the sps1 mutant. The results of scanning electron microscopy showed that when compared with the wild type, the inside and outside the central lemma of epidermal cells of sps1 was shorter and broader, and paraffin section showed that the smaller seed size of sps1 was caused by reduced cell length, increased cell width and cell number. There were significant changes in the expression of genes regulating rice grain size by controlling cell extension and division, such as the AFD1, SLG, HGW and GS3 significantly increased, the GW7 and GID1 significant reduced. Choose the single mutant strains in F₂ generation of Jinhui10/sps1 accord with the 3:1 separation ratio to conduct gene localization. And SPS1 was finally mapped on chromosome 7 with a 134 kb physical distance between markers sps1-3 and sps1-2. There are 19 annotated genes in the fine mapping region. Sequencing indicated that a single nucleotide substitution from A to T occurred in Os07g0616000, and a lysine was changed into termination codon, and then leaded to protein coding early termination in sps1. The root, stem, leaf, sheath and spike of the wild type and sps1 were analyzed by qPCR, and the results showed that the target gene was expressed in each organ of the plant, and the expression was highest in the plant stem. Bioinformatics analysis showed that SPS1 was a new allele of DEP2. The sensitivity of sps1 to the exogenous BR was reduced, and the expression of D1 was significantly reduced. It is speculated that SPS1/DEP2 may regulate the development of rice grain and plants by BR signaling pathway.【Conclusion】sps1 was a shorten panicle and seed mutant. SPS1 encoded an expression protein and is a new allele of DEP2, which regulated the development of rice grain and plant type by BR signaling pathway.

Morphological Characterization and Gene Mapping of a Panicle Apical Abortion Mutant (sipaa1) in Foxtail Millet

XUE HongLi, YANG JunJun, TANG Sha, ZHI Hui, WANG Rui, JIA GuanQing, QIAO ZhiJun, DIAO XianMin

Scientia Agricultura Sinica. 2018, 51(9):  1627-1640.  doi:10.3864/j.issn.0578-1752.2018.09.002

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【Objective】Panicle development determines crop yield, and panicle apical abortion is one of the major limitation that affects the grain yield of foxtail millet. Our study explored the genes and biological pathways related to panicle apical abortion in foxtail millet, which provided a theoretical basis for the genetic mechanism of foxtail millet panicle development.【Method】A foxtail millet panicle apical abortion mutant sipaa1, induced from Yugu1 by EMS treatment, was genetically identified. Agronomic traits of the mutant were investigated. The F₂ segregating population of sipaa1× SSR41 was used for gene mapping. Based on differential expressed levels of candidate genes in five different tissues of foxtail millet, some genes highly expressed in the panicle was identified. Transcriptome sequencing of Yugu1 and sipaa1 young panicle at booting stage was conducted to find differential expressed genes and to analyze the biological pathways.【Result】The mutant exhibited thinner panicle, shorter leaf, less spikelets per panicle, lower spikelet numbers, as well as lower 1000-grain weight, compared with wildtype Yugu1. The agronomic traits of mutants showed that mutant leaf length, leaf width, panicle length, panicle diameter, panicle weight per plant, spikelet number per panicle, grain weight per spikelet and 1000-grain weight were decreased 10.66%, 5.08%, 11.36%, 16.12%, 30.02%, 32.58%, 30.55%, 18.18%, respectively. Genetic analysis showed that segregating ratio of wild type to mutant plants were 3:1 in sipaa1×SSR41 F₂ generation, suggesting that the panicle apical abortion trait of sipaa1 was controlled by a single recessive nuclear gene. By map-based cloning, the candidate gene was mapped into a region between Indel markers In1-9.23 and In1-9.333 in chromosome 1, which contains a 100 kb interval. Combined with transcriptome sequencing, some candidate genes were found. Six candidate genes highly expressed in the panicle were further identified. Moreover, transcriptome sequencing showed that there were 2 768 up-regulated genes and 507 down-regulated genes between mutant and wild type. The differential expressed genes were mainly enriched in hormone signal transduction, external stress responses, plant-pathogen interaction.【Conclusion】Casual gene was limited into a 100 Kb region between Indel markers 1-9.23 and 1-9.333 on chromosome 1 of foxtail millet. Combined transcriptome sequencing and gene function analysis, we identified two candidate genes that are highly expressed in panicle, and reported to participate in plant floral development and stress responses. Panicle apical abortion may be regulated by hormone, stress responses, and programmed cell death pathway.

Fast Development of High Oleate Peanut Cultivars by Using Marker-Assisted Backcrossing and Their Evaluation

ZHANG ZhaoHua, WANG ZhiHui, HUAI DongXin, TAN JiaZhuang, CHEN JianHong, YAN LiYing, WANG XiaoJun, WAN LiYun, CHEN Ao, KANG YanPing, JIANG HuiFang, LEI Yong, LIAO BoShou

Scientia Agricultura Sinica. 2018, 51(9):  1641-1652.  doi:10.3864/j.issn.0578-1752.2018.09.003

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【Objective】 The objectives of this research were (i) to establish an effective backcrossing approach combined with marker-assisted selection (MAS) of peanut high oleate breeding, (ii) to transform the elite peanut cultivars with normal oleate (NO) into high oleate (HO) ones in three years.【Method】Four cross combinations were designed named as BO-01, BO-02, BO-03 and BO-04 by selecting the elite peanut cultivars ZH16, ZH21, QH551 and XH13 with AABB genotype as recurrent parents, while JH13 with aabb genotype was selected as the high oleate donor parent. Via 1 cross, 4 backcrosses and 1 self-crossing, BC₄F₂ progenies were obtained. According to the separation rule of backcross and high homology sequence of ahFAD2A and ahFAD2B, F₁ and backcross progenies（BC₁F₁-BC₄F₁）were genotyped by sequencing of PCR products, in which ahFAD2A and ahFAD2B were amplified in one PCR reaction with F0.7/R3 primer pairs. AaBb genotype progenies were selected and used as male parent in next generation backcross. Self-crossing progenies (BC₄F₂- BC₄F₃）were examined by KASP（kompetitive allele specific PCR）to obtain aabb genotype progenies. Main agronomic traits, quality characters and disease resistance of aabb genotype progenies were investigated. The genetic backgrounds of progenies with aabb genotype derived from ZH 16×JH13 were evaluated with SSR markers. 【Result】In summary, during 3 years, we obtained 10，5，6 and 8 high oleate progenies with aabb genotype in 4 cross combinations, respectively. By evaluating their agronomic traits, quality and bacterial wilt resistance, high oleate peanut cultivars were obtained, which were highly consistent with their corresponding recurrent female parent. ZJ019, ZJ109, ZJ160 and ZJ805 with oleic content as 82.54%, 79.85%, 79.22% and 78.94%, are regarded as high oleic cultivars of ZH16, ZH21, QH551 and XH13.【Conclusion】By using backcross, off-season multiplication for advancing generation and molecular marker selection, normal oleate peanut cultivars were successfully and quickly transited into high oleate ones within 3 years.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Responses of Extracellular Enzymes to Carbon and Phosphorus Additions in Rice Rhizosphere and Bulk Soil

LIU YuHuai, WEI XiaoMeng, WEI Liang, ZHU ZhenKe, GE TiDa, ZHANG YanJie, LU ShunBao, WU JinShui

Scientia Agricultura Sinica. 2018, 51(9):  1653-1663.  doi:10.3864/j.issn.0578-1752.2018.09.004

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【Objective】 Therefore, the objective of this experiment was to study the effects of exogenous nutrient input on soil microbial activity and phosphatase activity which was closely related to the phosphorus cycle in paddy soil, so as to clarify the responses of the phosphorus activity to carbon and phosphorus additions in rice rhizosphere and bulk soils. It is of great significance to guide the fertilization and management of rice soil and to realize the sustainable utilization of farmland. 【Method】 The typical phosphorus-limited rice soil in Hunan was selected for pot experiment. The treatments included: no carbon and phosphorus additions (CK), carbon addition (C), phosphorus addition (P), and carbon and phosphorus additions (CP). Acid phosphatases (ACP) and alkaline phosphatases (ALP) of the rhizosphere and bulk soils were determined by 96-microtiter plateau method. 4 soil phosphorus (CaCl₂-P, Citrate-P, Enzyme-P and HCl-P) were measured by biologically based phosphor (BBP) method to investigate the response of carbon and phosphorus addition and 4 soil phosphorus components based on the biological availability to soil enzyme activity.【Result】Compared with CK, the above-ground biomass of C, P and CP significantly increased by 29.76%, 84.03% and 87.94%, respectively (P＜0.05), and the underground biomass significantly decreased by 20.13% and increased by 57.49% and 56.53%, respectively (P＜0.05), which was consistent with the content of plant total phosphor (P＜0.05). The results showed that pH, the contents of NH₄⁺-N and Olsen-P in rhizosphere soil were significantly lower than those in bulk soil (P＜0.05). The content of microbial biomass phosphorus (MBP) in rhizosphere soil (except CP treatment) was significantly higher than that in bulk soil (P＜0.05). Carbon and phosphorus additions had a significant regulatory effect on 4 soil phosphorus components based on the biological availability in paddy rhizosphere and bulk soils (P＜0.05). Olsen-P and MBP had a significant negative correlation with ALP (P＜0.05), but no significant correlation with ACP, indicating it was obvious for microbes to utilize available nutrients. Phosphatase activity in bulk soil was mainly affected by Olsen-P and MBP, as well as CaCl₂-P and Citrate-P content, while soil water content, pH, NH₄⁺-N and root biomass, as well as HCl-P and Enzyme-P contents mainly affected phosphatase activity in rhizosphere soil. 【Conclusion】 The application of P and CP addition could improve the microbial activity of phosphorus-limited rice soil, increase the biomass significantly, and improve the rhizosphere microbes benefit and amendment the soil environment as well as benefit the health of paddy field ecosystem.

Effects of Straw Returning in Matching and Nitrogen Fertilizer on Crop Yields, Water and Nitrogen Utilization Under Wheat-Maize Multiple Cropping System

YANG ChenLu, LIU LanQing, WANG WeiYu, REN GuangXin, FENG YongZhong, YANG GaiHe

Scientia Agricultura Sinica. 2018, 51(9):  1664-1680.  doi:10.3864/j.issn.0578-1752.2018.09.005

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【Objective】This research was conducted to explore the response of crop production under wheat-maize multiple cropping system in Shaanxi Guanzhong region to straw returning and appropriate nitrogen fertilizer application conditions, so as to provide theoretical basis for realizing local crop yield increase and efficient utilization of resources. 【Method】 A five-year field experiment was persistently performed in Yangling of Shaanxi Province from October 2011 to October 2016. A split plot design was developed in this experiment, with different straw returning patterns (straw returning (S) and no straw returning (S0)) as main treatments and different fertilizer applications (farmer actual fertilization (F1), 20% fertilizer reduction (F0.8) and no fertilization (F0)) as sub-treatments. Crop yield, water and nitrogen utilization of winter wheat and summer maize under different treatments were measured and analyzed, respectively. 【Result】 The interactions of straw returning and nitrogen applications on biomass, yield and yield components, water and fertilizer utilization of wheat and maize were significant or highly significant. Compared with no straw returning, the content of soil organic matter, alkali-hydrolyzable nitrogen, available phosphorus and available potassium under the condition of straw returning significantly increased 6.0%-13.9%, 8.2%-34.1%, 3.4%-4.7%, 3.3%-10.3%, respectively, and the soil water storage in 0-100 cm soil layer before sowing and after harvesting of winter wheat and summer maize significantly increased 5%-11%, 12%-15% in wheat and 4%-9%, 11%-17% in maize, respectively. Compared with no fertilization, the soil nutrient content under fertilization was significantly increased, and the soil water storage of both two crops with straw returning under the condition of fertilization was significantly increased. Compared with no straw returning, the yield of grain in four continuous wheat growing seasons (2012-2016) and five continuous maize growing seasons (2011-2016) under the condition of straw returning, respectively, increased 4%-6%, 5%-10%, 7%-10%, 8%-12% in wheat and 1%-2%, 3%-6%, 4%-7%, 5%-8%, 3%-7% in maize significantly; Water use efficiency (WUE), respectively, increased 4%-7% in wheat and 8%-11% in maize significantly; Nitrogen partial factor productivity (PEPN) and agronomic efficiency (AEN) of both two crops were increased significantly. Compared with no fertilization, the yield of wheat and maize under the condition of fertilization was increased significantly, and was highest in F1 treatment, which was significantly increased by 30%-38%, 29%-33%, respectively, in wheat and 21%-25%, 19%-22%, respectively, in maize comparing with F0 treatment under two straw returning level; WUE of both two crops was increased significantly, but under S0 level, the F1 treatment was the highest, and the F0.8 treatment was the highest under S level. Compared with farmer actual fertilization, PEP_N and AE_N of both two crops were significantly increased under 20% fertilizer reduction, and the highest SF0.8 treatment was significantly higher 31%, 30% in wheat and 30%, 31% in maize than the lowest S0F1 treatment, respectively. In terms of economic benefits, annual average net income of wheat and maize under straw returning condition was 808-1 258 yuan and 733-1 212 yuan higher than that of no straw returning, and compared with no fertilization condition, annual average net income of two crops was increased, and the F0.8 treatment gained the most. Meanwhile, two crops both showed the trend of SF0.8＞ SF1＞S0F0.8＞S0F1＞SF0＞S0F0 in five years, of which SF0.8 treatment increased 3 052 yuan in wheat and 2 145 yuan in maize respectively compared with the CK. 【Conclusion】 Generally, comparing with local farmer actual fertilization, long-term straw returning in matching with 20% reduction fertilization not only ensured the crop yield in a high level, but also improved utilization of water and nitrogen significantly. Based on the efficiency of water and nitrogen utilization, crop yield and economic benefit, the effect of SF0.8 treatment was the optimal in all treatments.

Key Research Priorities for Multiple Cropping Systems

WU WenBin, YU QiangYi, LU Miao, XIANG MingTao, XIE AnKun, YANG Peng, TANG HuaJun

Scientia Agricultura Sinica. 2018, 51(9):  1681-1694.  doi:10.3864/j.issn.0578-1752.2018.09.006

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To feed the China’s growing population, more food needs to be produced using currently available cropland. Further expansion of cropland seems to be unlikely as it largely conflicts with biodiversity conservation, greenhouse gas emission mitigations, and hydrological changes. Crop yield growth is also confronting with large challenges as it becomes more difficult to sustain further yield increase as farmers’ yields approach the potential threshold. Increasing cropping intensity may thus provide another promising opportunity to increase food production. An increase in cropping intensity by increasing the number of crops per cropping cycle or intercropping with other crops can increase the frequency of harvests each year, resulting in increased food supplies without additional cropland expansion. This paper, from the perspective of agricultural land systems, proposed the overall study framework of multiple cropping systems and provided an overview of current research progresses in the three key research priorities. It was indicated that: (1) Multi-faceted patterns and processes of multiple cropping systems were the basis for subsequent analysis. It should consider not only the quantity, spatial distribution, regional difference of potential or actual multiple cropping systems, but also the cropping intensity gaps so as to expand the harvest areas by closing these gaps. (2) Understanding of the effects of multiple cropping systems on socio-economic and biophysical systems was the core task. Currently, most of studies were focused on the positive contribution of multiple cropping systems to food production, and less attention was paid to its impacts on eco-environment. Interdisciplinary approaches and cross-scale integration are critically necessary for better understanding the complex effects and feedbacks of multiple cropping systems on regional resource allocation, socio-economic development and eco-environment health. (3) How to sustainably increase the multiple cropping indexes was facing great challenges. It was important to link the multiple cropping systems with other parallel systems to understand their synergies and trade-offs, in order to build up a sustainable pathway for increasing future cropping intensity. All these solutions would substantially promote the interdisciplinary integration and would contribute to better understand the coupled human-environment interactions across time, space and scales.

PLANT PROTECTION

Construction of Citrus leaf blotch virus Infectious cDNA Clone by Yeast Homologous Recombination System

CUI TianTian, YAN JianHong, BIN Yu, LI ZhongAn, ZHOU ChangYong, SONG Zhen

Scientia Agricultura Sinica. 2018, 51(9):  1695-1705.  doi:10.3864/j.issn.0578-1752.2018.09.007

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【Objective】 The objective of this study is to construct infectious cDNA clone of Citrus leaf blotch virus (CLBV) isolated from China and lay a foundation for studies on the pathogenesis of CLBV at the molecular level.【Method】Using GeneArt® pYES1L Vector as a template, a 2.1-kb fragment containing the yeast replication origin and Trp-1 gene were amplified by primer pair PYES2117F/PYES2117R. Plasmid of a binary vector DK1317-2 was digested with Sac II. The amplified and digested productions containing expected fragments were then purified with the Gel Extraction Kit and subjected to In-Fusion cloning. In this way a ternary shuttle vector pCY was obtained. Using Potato virus X (PVX) infectious clones as a template, pCY-PVX-F and pCY-PVX-R as primers, the full length cDNA of PVX genome was amplified. Plasmid pCY was digested with restriction endonuclease Stu I and Sma I. The fragment and the linearized vector obtained above were co-transformed into yeast YPH501 by lithium acetate conversion method, and the full-length cDNA clone of PVX genome was obtained by homologous recombination. The infectivity of the resulting constructs was verified by Agrobacterium tumefaciens mediated inoculation on N. benthamiana, and a rapid cloning system based on homologous recombination in yeast cell was established for construction of viral infectious clone. On this basis, two fragments covering the full-length genome of CLBV-HBYD were amplified, and CLBV-1 and CLBV-2 were obtained. The homologous recombination was then performed to assemble CLBV-1, CLBV-2 and pCY vector fragments using the cloning system established. Then, N. benthamiana and Jincheng (C. sinensis) was inoculated with A. tumefaciens carrying the recombinant plasmid pCY-CLBV. RT-PCR and Northern blot were used to detect the inoculated seedlings.【Result】A ternary yeast-E. coli-A. tumefaciens shuttle vector, pCY, was constructed based on DK1317-2 and pYES1L. The vector is 10 347 bp in length and contains the replication elements of three bacteria, and can be stably replicated in yeast, A. tumefaciens, and E. coli. Using this vector, 16 full-length cDNA clones of CLBV-HBYD genome were obtained through homologous recombination in yeast cell. One clone was randomly selected and sequenced (GenBank: MG572236). Sequence analysis showed that CLBV-HBYD is 8 747 nt in length and encodes 3 open reading frames (ORF). ORF1, ORF2 and ORF3 is 5 889, 1 089 and 1 092 nt, respectively. The nucleotide sequence identity between CLBV-HBYD and 9 isolates of CLBV ranged 79%-98%, in which the highest identity was 98% with EU857540 isolate from citrus, while only 79% was seen when compared with kiwifruit isolates JN983454, JN983455, JN983456 and JN900477. In the phylogenetic tree generated by MEGA 6 software, CLBV-HBYD clustered with other citrus isolates into the same cluster. Sixteen CLBV full-length cDNA clones were inoculated on N. benthamiana and Jincheng by A. tumefaciens mediated inoculation, and pCY empty vector was used as a negative control. After 20 days of inoculation, RNA was extracted for RT-PCR detection. The results showed that CLBV specific genes were detected in plants inoculated with 11 full-length cDNA clones of CLBV-HBYD, respectively. Five set of RT-PCR positive plants were randomly selected for further Northern blot. The results showed that specific bands of CLBV were detected by Northern blot in plants inoculated with pCY-CLBV 1, 2, 3, 14, 15, whereas no gene was detected in the control samples, indicating that these clones were infectious clones.【Conclusion】A rapid cloning system based on a ternary yeast-E. coli-A. tumefaciens shuttle vector pCY and homologous recombination in yeast was constructed, and an infectious cDNA clone of Chinese isolate of CLBV was developed for the first time.

Establishment of RT-LAMP Assay for Detection of Apple chlorotic leaf spot virus (ACLSV)

ZHANG ShuangNa, LI ZhengNan, FAN XuDong, ZHANG ZunPing, REN Fang, HU GuoJun, DONG YaFeng

Scientia Agricultura Sinica. 2018, 51(9):  1706-1716.  doi:10.3864/j.issn.0578-1752.2018.09.008

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【Objective】The objective of this study is to establish a method which uses reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology, and to detect Apple chlorotic leaf spot virus (ACLSV) simply and quickly. 【Method】Three sets of specific primers were designed based on conserved region of ACLSV genomes. Each set of primers includes a pair of outer primer (F3/B3) and a pair of inner primer (FIP/BIP). One feasible set of primers was selected for the RT-LAMP reaction. RT-LAMP reaction system was optimized, that is, the concentration of Mg²⁺ (0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mmol·L^-1), dNTPs (0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 mmol·L^-1), Betaine (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mol·L^-1), FIP/BIP (0.8, 1.2, 1.6, 2.0, 2.4 µmol·L^-1) and F3/ B3 (0, 0.1, 0.2, 0.3, 0.4 µmol·L^-1), respectively. The RT-LAMP reaction condition was optimized, using optimized reaction system, reaction temperature was set to 65, 63, 61, 59, 57℃ and reaction time was set for 90 min. In the process of primers screening and optimizing the reaction system, fluorescent pigment was added to the reaction system and real-time PCR instrument was used. The whole process was detected in real-time by fluorescence signal accumulation. Finally, the amplification curve was used to analyze the reaction result. the specificity detection of RT-LAMP was tested by using different RNA templates of infected leaves from ACLSV, Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple mosaic virus (ApMV). To assess the detection sensitivity, 10⁰, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 diluents of original RNA were used as templates of RT-LAMP. To evaluate the application value of this method, 23 apple leaf samples were collected randomly in the field, the optimized RT-LAMP and RT-PCR were used to detect the samples, SYBR GreenⅠwas added to visualize the detection. 【Result】 The RT-LAMP method to detect ACLSV was established. The optimized detection system was 6.0 mmol·L^-1 Mg²⁺, 1.2 mmol·L^-1 dNTPs, 0.2 mol·L^-1 Betaine, 1.6 µmol·L^-1 FIP/BIP and 0.2 µmol·L^-1 F3/B3 primer. The optimum reaction conditions were 59℃ and 60 min. In the specificity test, only the ACLSV test result was positive, the controls were all negative. In the sensitivity test, the 10^-3 RNA dilution could be detected by RT-LAMP method. It was 100 times higher sensitivity than RT-PCR method. The positive rate of RT-PCR of randomly selected 23 apple leaf samples was 52.2%, and RT-LAMP was 65.2%. Detection rate of RT-LAMP was higher than that of RT-PCR. 【Conclusion】The established ACLSV RT-LAMP detection method is simple, quick, sensitive and low cost. It can be applied in field investigation, seeding breeding and customs quarantine.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Spectral Characteristics and Quantitative Prediction of Soil Water Content under Different Soil Particle Sizes

LU YanLi, BAI YouLu, WANG Lei, YANG LiPing

Scientia Agricultura Sinica. 2018, 51(9):  1717-1724.  doi:10.3864/j.issn.0578-1752.2018.09.009

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【Objective】Hyper spectral technology is more and more widely applied in monitoring soil properties. Soil Water Content (SWC) is a key parameter of soil properties. This paper aimed to make clear the spectral response of soil moisture under different soil roughness to predict quantitatively soil water content, and further to provide the basis for rapid monitoring of farmland moisture and other soil properties. 【Method】 Soil samples were sieved through different mesh sizes to classify into different particle sizes, and then set different moisture levels. The spectral reflectance of those samples were compared, and the quantitative prediction models of soil water content were established by Partial Least Squares Regression (PLSR) method. 【Result】The results showed that the spectral reflectance decreased with the increase of soil water content, and the difference became bigger as the increase of wavelength and decrease of soil water content. The absorptions got deeper in 1 400 nm and 1 900 nm with the increase of water content. In those soil samples passed through a sieve with an aperture of 0.15 mm (denoted as D-1), the spectral reflectance increased in 350-1 240 nm and then decreased after 1 240 nm when the water content was more than 40%. Compared with the model constructed from all samples with different sizes, models from the same size were improved in predicting accuracy and stability: the smaller the particle size was, the better the prediction effect and stability of the predicting model were. The RMSE (root mean square error) and R2 of the optimal model were 4.13% and 0.90, respectively. Additionally, normalization of spectral data reduced the influence of noise, and improved the predicting accuracy and stability of the model. 【Conclusion】The spectral generally decreased with soil water content increasing, but soil with small size showed opposite in 350-1 240 nm when the moisture content was greater than 40%. The predicting model for soil water content was improved as size getting smaller and sample number involved getting larger, and the spectral data normalization also improved predicting accuracy and stability of model.

Effects of Nitrogen Fertilizer and Dicyandiamide Application on Tomato Growth and Reactive Nitrogen Emissions in Greenhouse

YIN Xing, ZHANG LiJuan, LI BoWen, LIU WenJu, GUO YanJie, LI YuTao

Scientia Agricultura Sinica. 2018, 51(9):  1725-1734.  doi:10.3864/j.issn.0578-1752.2018.09.010

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【Objective】Effects of nitrogen (N) fertilizer and inhibitor (dicyandiamide, DCD) application on tomato yields, quality and reactive nitrogen loss were studied under field condition, clearing the function and nitrification inhibitory effect of DCD in greenhouse vegetable production systems, to provide scientific basis for reducing nitrogen fertilization and increasing efficiency, and prevention and control of pollution. 【Method】A field experiment was conducted in the Yongqing County of Hebei Province and the test crop was tomato. The experiment consisted of 5 N fertilization treatments with three replicates: control treatment (N0), conventional N fertilization rate (Con), conventional N fertilization plus nitrification inhibitor (Con+DCD), optimal N fertilization rate (Opt), and optimal N fertilization plusnitrification inhibitor (Opt+DCD). By field-situ tracking method, soil inorganic nitrogen, N₂O emissions, ammonia volatilization loss and other indicators were measured during the top dressing of greenhouse tomato; N₂O samples were measured using a gas chromatograph, and soil inorganic nitrogen samples were analyzed by using a continuous flow analytical system; ammonia volatilization samples were measured by boric acid absorption- standard acid; SAS software were applied on the yield, quality and various indicators of different treatments for variance analysis.【Result】Nitrogen fertilizer combined with DCD could increase tomato yield, namely, the Con+DCD and Opt+DCD increased the yields by 20.2% and 2.4% compared with the Con and Opt, respectively. Tomato yield of Con+DCD was significantly higher than that of the Con treatment. Simultaneously, the NAE and PFP for the Con+DCD and Opt+DCD were significantly higher than the Con and Opt. Compared with the Con and Opt, the NAE for the Con+DCD and Opt+DCD was increased by 176.7% and 22.3%, respectively. In addition, the NO₃^--N content in tomato was significantly decreased after combination of nitrogen fertilizer and DCD, thus, compared with Con and Opt, Con+DCD and Opt+DCD decreased the NO₃^--N content by 28.6% and 19.3%, respectively. There was no significant difference among other quality indicators. The NO₃^--N accumulation at 0-100 cm soil depth under Con+DCD and Opt+DCD treatments were 607.1kg·hm^-2 and 441.8 kg·hm^-2, which were 14.3% and 15.7% lower than NO₃^--N accumulation under Con (708.4 kg·hm^-2) and Opt (524.2kg·hm^-2), respectively. N₂O emission flues and ammonia volatilization rate reached peak values on second and third day after fertilization. Overall, N₂O emissions and NH₃ volatilization loss were reduced under the DCD treatment. Compared with the Con and Opt, N₂O accumulative emission and accumulative N loss by NH₃ volatilization for the Con+DCD and Opt+DCD decreased by 51.2%, 75.4% and 17.2%, 21.9%, respectively. 【Conclusion】Under the experimental conditions, combination of nitrogen fertilizer and DCD increased tomato yields, NAE and PFP, and decreased the NO₃^--N accumulation at 0-100 cm depth, emission flue of N₂O and ammonia volatilization loss. Opt+DCD showed the best effect among the treatments. Therefore, reducing Nitrogen and combined application of DCD is a scientific and effective fertilizer management in greenhouse tomato production.

Effects of Vanillic Acid on Seed Germination, Seedling Growth and Rhizosphere Microflora of Peanut

HUANG YuQian, YANG JinFeng, LIANG ChunHao, CHEN ShenPingYi, LIU XinYu, GENG KeRui, YAO YuChen, ZHANG Yu, HAN XiaoRi

Scientia Agricultura Sinica. 2018, 51(9):  1735-1745.  doi:10.3864/j.issn.0578-1752.2018.09.011

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【Objective】 Experiments were carried out to investigate the autotoxicity of vanillic acid and its effect on seed germination and seedling growth of peanut, so as to reveal the response rules of rhizosphere microbes to the autotoxic substances in peanut growth period.【Method】Peanut (Arachis hypogaea L. cv Fuhua 12 150GY ) was used in this study, and petri dish method, nursery pot and pot culture experiment were adopted to explore the effect of vanillic acid on the peanut seed germination, seedling growth and rhizosphere microflora. Six vanillic acid application treatments in petri dish experiment were set with application of different vanillic acid amounts: 0, 0.01, 0.03, 0.05, 0.07, 0.09 mmol·L^-1, five treatments in nursery pot experiment were 0, 0.01, 0.03, 0.05, 0.07 mmol·L^-1, and five treatments in pot culture experiment were 0, 0.01, 0.03, 0.05, 0.07 mg·kg^-1 dry soil.【Result】(1) The seed germination ratio, germination energy and germination index were lower than CK after treatment with different concentrations of vanillic acid. When the concentration of vanillic acid was 0.09 mmol·L^-1, the seed germination ratio, germination energy and germination index respectively decreased by 39%, 66.3% and 55.9% compared with CK, and the response index (RI) reached the maximum. (2) The root length, plant dry weight, chlorophyll content, net photosynthetic rate and stomatal conductance were lower than CK after treatment with different concentrations of vanillic acid. When the concentration of vanillic acid was 0.07 mmol·L^-1, all above indexes decreased by 37.3%, 40.0%, 19.0%, 53.9% and 49.1%, respectively, compared with CK, and the response index (RI) reached the maximum. Conversely, the intercellular CO₂ concentration was the opposite of the above indexes, it went up with the increase of the concentration of vanillic acid. When the concentration of vanillic acid was 0.07 mmol·L^-1, the intercellular CO₂ concentration increased by 46.1% compared with CK. (3) when the concentration of vanillic acid ≥0.03 mmol·L^-1, the total absorption area, active absorption area and root activity were lower than CK, while the MDA content were higher than CK. When the concentration of vanillic acid was 0.07 mmol·L^-1, all above indexes respectively decreased by 22.4%, 54.2% and 40.6% compared with CK, the MDA content increased by 43.3%. (4) The number of rhizosphere actinomycetes were markedly reduced with the increase of vanillic acid concentration at the early stage of peanut, and the differences between the treatments were not significant at the late stage of peanut. The number of bacteria in different treatments were not significant difference at the early stage of peanut, and it substantially reduced with the increase of vanillic acid concentration at the late stage. A high concentration of vanillic acid (0.07 mg·kg^-1 dry soil) had inhibitory effect on rhizosphere fungal growth, and low concentration of vanillic acid (0.01 mg·kg^-1 dry soil) had a promoting effect of fungal growth. 【Conclusion】 Vanillic acid had a significant autotoxinc-effect, which inhibited the photosynthesis of peanut seedling, reduced the root activity and promoted seedling leaf malondialdehyde. In addition, the number of rhizosphere bacteria and actinomycetes decreased after treated with vanillic acid, indicating that vanillic acid inhibited the growth of rhizosphere bacteria and actinomycetes; while it promoted the growth of rhizosphere fungi at low concentration but inhibited the growth at high concentration.

HORTICULTURE

Development of Fertility-Restored BC₃ Progenies in Ogura CMS Chinese Kale and Analysis on Gene Transmission Rate of Rfo and Genetic Background

YU HaiLong, LI ZhiYuan, YANG LiMei, LIU YuMei, ZHUANG Mu, Lü HongHao, LI ZhanSheng, FANG ZhiYuan, ZHANG YangYong

Scientia Agricultura Sinica. 2018, 51(9):  1746-1757.  doi:10.3864/j.issn.0578-1752.2018.09.012

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【Objective】 Development of Ogura cytoplasmic male sterility (Ogura CMS) restorer lines is an effective way to utilize Ogura CMS germplasm resources. In order to obtain the Ogura CMS fertility-restored lines in Brassica oleracea, BC₂ fertility-restored individuals 15Q23 were backcrossed with Chinese kale parent, and the transmission rate (TR) of Rfo restorer gene, genetic background, seed setting and ploidy were investigated in the BC₃ Rfo-positive progenies, which will accelerate the creation the Ogura CMS fertility restorer lines in B. oleracea. 【Method】 The BC₂ fertility-restored individual 15Q23 with better fertility performance was chosen as male parent to backcross with Ogura CMS Chinese kale 15Y102. The BC₃ progenies were produced and screened by the Rfo specific markers. The TR of Rfo in BC₃ generations was calculated. Morphological identification, fertility observation, seed setting ability and ploidy evaluation were performed to select BC₃ Rfo-positive individuals with better seed setting and genetic background similar to the parent Chinese kale; these individuals were then used as pollen donors to develop the BC₄ generation. The TR of Rfo and ploidy of the Rfo-positive individuals in BC₄ generations were evaluated. 【Result】 The pollen viability and seed setting of 15Q23 differed at different flowering periods, with an average of 0.07 seeds per pod (7%). The BC₃ generations individuals were screened by Rfo-specific marker and the Rfo could be transmitted stably. The genetic similarity coefficients between BC₃ individuals and Chinese kale 15Y102 were 0.81-0.92, higher than that of 15Q23 (0.73), indicating that the genetic background of BC₃ individuals was closer to the parent Chinese kale. These results were further confirmed by the clustering result of morphological markers. Morphological observation revealed that the BC₃ individuals were very similar to the parent Chinese kale 15Y102, whereas the growth vigor of BC₃ individual was higher than that of the parent Chinese kale. Ploidy identification showed that most of the BC₃ individuals were still close to tetraploid. During the flowering stage, all of the BC₃ Rfo-positive individuals were fertility-restored and the pollen viability of them was different among different BC₃ individuals. The individuals 16Q1-4, 16Q1-7, 16Q1-10 showed better fertility performance during the whole flowering period, and their pollen viability was above 75%. The individuals with ＞50% pollen viability were chosen as pollen donors to backcross with Chinese kale and their seed setting was calculated. Compared with other BC₃ Rfo-positive individuals, the seed setting of individual 16Q1-4 and 16Q1-10 was 15% and 9%, respectively, much higher than other BC₃ Rfo-positive individuals, and significantly higher than that of 15Q23 (7%, P＜0.05). Furthermore, the Rfo could be transmitted in BC₄ generation and the TR of Rfo was nearly 33%. Ploidy identification was performed among the 24 BC₄ Rfo-positive individuals, which were derived from 16Q1-4. The results indicated that the ploidy differed in the 24 BC₄ Rfo-positive individuals and the peaks of G₀/G₁ period in three individuals were close to that of parent Chinese kale. 【Conclusion】 The interspecific hexaploid hybrid was further backcrossed with Chinese kale to produce the BC₃ and BC₄ generations. The results indicated that the Rfo can be stably transmitted. Two individuals, 16Q1-4 and 16Q1-10, morphologically similar to parent Chinese kale 15Y102, have been successfully created with closer genetic background to Chinese kale compared with BC₂ pollen donor 15Q23, and significantly improved seed setting.

Effects of Different Nitrogen and Potassium Fertilizing Amount on Nutrition Absorption, Nutrition Distribution and Yield of Muskmelon

KANG LiYun, CHANG GaoZheng, GAO NingNing, LI XiaoHui, LI HaiLun, LIANG Shen, XU XiaoLi, ZHAO WeiXing

Scientia Agricultura Sinica. 2018, 51(9):  1758-1770.  doi:10.3864/j.issn.0578-1752.2018.09.013

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【Objective】To provide a theoretical basis for the rational application of nitrogen and potassium fertilizers with plastic film mulching in high yield cultivation environment, the effects of different nitrogen and potassium fertilizers in the condition of the medium fertile soil on nutrient absorption, distribution and yield of muskmelon were studied.【Method】An early maturing and thick-skinned muskmelon variety ‘RX8’ (TC620-8-56×TA11-1) with plastic film mulching cultivation in greenhouse was used for our study. Five treatments (N2K2, N2K1, N1K2, N1K1 and NK) were performed to investigate the influence of different fertilizing amount of nitrogen and potassium on nutrient absorption, distribution and yield of muskmelon. The accumulation of dry matter and nitrogen, phosphorus and potassium were measured in different organs at the vining, fruit-set and mature stages. Furthermore, we also measured the production of muskmelon at maturity. 【Result】The results indicated application of different nitrogen and potassium fertilizers on the accumulation of dry matter and nitrogen, phosphorus and potassium in muskmelon posed a similar variation trend, and had an increasing level with the growth. Applied different amount of nitrogen and potassium fertilizers changed the accumulation amount of dry matter and nitrogen, phosphorus and potassium at different growth stages, but did not change the accumulation trend. From the characteristics of dry matter accumulation and distribution, the muskmelon was mainly vegetative growth at the vining stage, and the dry matter accumulation in root, stem and leaf had a high level under NK treatment in this stage. The accumulation of dry matter in stem and leaf under NK treatment increased averagely by 17.8% and 16.0% respectively, which was significantly higher than that under other treatments (P＜0.05). The accumulation of dry matter increased under different nitrogen and potassium fertilizers during fruit development stage, the distribution coefficient was about 0.62 to 0.66 at mature stage. The dry matter accumulation and its distribution coefficients in fruit increased averagely by 31.9% and 4.27% under NK treatment, respectively, which was significantly higher than that of other treatments (P＜0.05). As for the characteristics of the nutrient accumulation and distribution, potassium was the top demand of muskmelon, followed by nitrogen and phosphorus. The uptake amounts of nitrogen, phosphorus and potassium of muskmelon were 4 160.4 mg, 1 394.8 mg, 7 874.2 mg/plant at mature stage, respectively. The amounts of nitrogen, phosphorus and potassium uptake had a relatively higher level under NK treatment than that of other treatments at the vining, fruit-set and mature stages all the time (P＜0.05). The distribution coefficients of nitrogen, phosphorus and potassium in fruit at mature stage under NK treatment had a relative high level, of which increased by 9.10% and 9.81% respectively and was higher than other treatments in maturity fruit (P＜0.05). The yield and yield components, including the difference of the fruit vertical and transverse diameter, shape index, average weight and yield of high potassium and low potassium treatments did not reach 5% significant level under the same nitrogen level condition. The fruit vertical and transverse diameter, average weight and yield of high nitrogen-treated were higher than those of low-nitrogen treatment, with an average increase of 4.81%, 6.04%, 19.8% and 20.5% respectively under the same potassium supply. And the differences of these indexes except for vertical diameter reached a significant level of 5%. On the whole, the fruit vertical and transverse diameter, average weight and yield of muskmelon treated with NK were the highest, and average weight and yield were significantly higher than those of other treatments (P＜0.05), with an average increase of 21.6% and 22.1%, respectively.【Conclusion】This study demonstrated that the optimum fertilizing amount of nitrogen and potassium with plastic film mulching for muskmelon growth in the condition of the medium fertile soil was 200 kg·hm^-2 and 300 kg·hm^-2, respectively, which was beneficial to improve the ability of nutrient absorption and promote the absorption and distribution of the nutrient to the fruits

Cloning and Functional Verification of AINTEGUMENTA Gene in Chrysanthemum

WEN LiZhu, SUN Xia, FAN HongMei, GUO YunHui, YU YuanYuan, REN Hong, WANG WenLi, ZHENG ChengShu

Scientia Agricultura Sinica. 2018, 51(9):  1771-1782.  doi:10.3864/j.issn.0578-1752.2018.09.014

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【Objective】 To understand the role of AINTEGUMENTA gene in chrysanthemum inflorescence development, we cloned AINTEGUMENTA gene from chrysanthemum, analyzed its sequence, and characterized its temporal and spatial expression pattern. We further analyzed the impact of AINTEGUMENTA silence on inflorescence development and its possible regulation mode. This study was carried to reveal the potential mechanism of AINTEGUMENTA in governing inflorescence development, which in turn can provide a theoretical basis for chrysanthemum inflorescence diameter adjustment. 【Method】 The chrysanthemum AINTEGUMENTA gene was cloned by RACE method and its sequence was analyzed with DANMAN software. Its expression in different developmental stages and organs of chrysanthemum was detected by real-time fluorescent quantitation PCR. The plasmid of 35S::CmANT-GFP was constructed for the subcellular localization. The silence vector of TRV2-CmANT was constructed to infect chrysanthemums. The statistics of phenotypic changes in CmANT silenced chrysanthemums were analyzed via SPSS software. The ray florets petal epithelial cells were observed with optical microscope. The expression of CmANT and related genes were detected by real-time fluorescent quantitation PCR. 【Result】 The full-length of CmANT was cloned from chrysanthemum. It encodes 540 amino acids and contains two AP2 conserved function domains and VYL modification sites. The theoretical isoelectric point of CmANT is 7.39 and its molecular weight is 60.4 kD. The phylogenetic tree composed of ANT proteins from various plants species showed that CmANT and AaANT had been grouped together. The real-time fluorescent quantitation results showed that: (1) CmANT was expressed most in floral buds, followed by roots, stems and leaves. (2) Gene expression in different parts of inflorescence indicated that CmANT was expressed the most in ray florets followed tubular florets and the lowest in the sepal. (3) The expression of CmANT declined during the development of ray florets. (4) The expression of CmANT under 2,4-D treatment increased between 3 h to 6 h. WoLF PSORT prediction and 35S::CmANT-GFP fusion protein localization in onion epithelial cells indicated that CmANT protein was located in cell nucleus. Compared with the control, the mean inflorescence diameters of chrysanthemums in TRV-CmANT-1 and TRV-CmANT-2 lines were decreased by 18.93% and 27.47% respectively and the numbers of ray florets were decreased by 11.39% and 14.66% respectively, among which the difference between TRV-CmANT-2 lines and the control was statistically significant (P＜0.05 ). The numbers of tubular florets were decreased by 14.55% and 36.56%, the mean length of ray florets in top inflorescences was decreased by 34.17% and 54.68%, and the width was decreased by 24.05% and 10.13% respectively in TRV-CmANT-1 and TRV-CmANT-2 lines compared with the control, the mean fresh weight of leaves was decreased by 13.19% and 21.98% respectively in TRV-CmANT-1 and TRV-CmANT-2 lines and the correlation between leaf fresh weight and tubular floret number was significant (P＜0.05 ). The microscopic observation of epithelial cells in ray floret petals indicated that the cell length and width of petals in the silenced lines and the control had no visible differences. The expression of CmLAX3 in two silenced chrysanthemums lines were 1.8 and 1.78 times higher than the control, respectively. Meanwhile the expression of CmCYCD3 was decreased by 32.28% and 38.19% in two silenced lines and the expression of CmXTH4 and CmEXPA1 also decreased at most developmental stages. 【Conclusion】 Due to the phenotypic changes and CmANT expression pattern in silenced chrysanthemums lines, we speculated that the silence of CmANT might relieve the repression on CmLAX3, facilitate the transport and accumulation of auxins, indirectly repress the activation of CmCYCD3, limit the cell division, and cause the decrease of cell number, combined of which would eventually lead to the smaller organ sizes in silenced chrysanthemums lines.

FOOD SCIENCE AND ENGINEERING

The Changes of Protein Components and Subunits in Process of Mung Bean Germination

ZHAO TianYao, ZHANG YaHong, JIN Tao, KANG YuFan

Scientia Agricultura Sinica. 2018, 51(9):  1783-1794.  doi:10.3864/j.issn.0578-1752.2018.09.015

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【Objective】 In order to provide the scientific basis for processing and utilization of mung bean protein, the dynamic changes of mung bean isolated protein, albumin, globulin, gluten and gliadin in different germination stages were investigated.【Method】The isolated protein of mung bean were extracted by isoelectric point precipitation method, and the albumin, gluten, globulin and gliadin of mung bean were prepared according to Osborne classification. The content of each kind of proteins and the activity of protease were determined in different germination period. The changes of composition and quantity of protein subunit before and after germination were further analyzed by SDS-PAGE electrophoresis.【Result】With the extension of germination time, the contents of mung bean isolated protein increased first and then decreased. The Mung bean isolated protein was increased by 9.4% compared to non-germinated mung bean. The Mung bean albumin content was decreased after the germination, which the minimum value was 20.47 mg·g^-1. The globulin content was increased first and then decreased with the increasing of germination time, with the minimum was observed at 0 h and the highest at 48 h after germination. However, the gliadin content was not influenced by the germination. There was no significant difference in the gluten content of mung bean between the 12-36 h and 48-72 h germination, but the content is still higher than non-germinated seed. Mung bean protease activity increased within 36 h germination, and began to decline at 72 h. Analysis of SDS-PAGE electrophoresis and optical density scanning results showed that there were 7 bands (Ⅰ-Ⅶ) in the mung bean isolated protein, and the relative content of each band decreased continuously. After the germination, the molecular weight of 25 kD to 66 kD proteins gradually decreased. The content of 18 kD and 25 kD isolated proteins increased within 72 h germination, and almost only remained Ⅳ after 96 h of germination. Mung bean albumin mainly consisted of four bands (Ⅰ-Ⅳ) with molecular weights of 61.56, 48.99, 29.88 and 20.42 kD, respectively. During the germination process, the relative content of Band I decreased from 18.4% at 0 h to 16.4% at 60 h, and disappeared at 72 h after germination. Although the Band II existed during the whole germination, the relative content of Band II was decreased continuously from 51.1% to 27.0%. Bands Ⅲ and Ⅳ decreased continuously with the germination, and disappeared after 72h of germination. What’s more, the relative contents of subunit bands with a molecular weight of 18-25 kD increased at 24-60 h and disappeared after 72 h of germination. Mung bean globulin mainly consists of five bands (Ⅰ-Ⅴ) with molecular weights of 66, 61, 50, 32 and 26 kD, respectively. The relative content of subunit bands I and II were disappeared after 96 h of germination. While the relative content of band Ⅲ increased gradually from 34.4% to 41.8% during 0 to 60 h of early stage of germination. Then the band Ⅲ content decreased rapidly after 72 h of germination and measured only 10.8% at 96 h. In the meantime, the relative contents of subunit Band Ⅳ and Ⅴ was not observed after 84 h germination. Subunit bands with a molecular weight of 18-25 kD increased during 24 to 60 h of germination, and then degraded or even disappeared with the germination process.【Conclusion】Appropriate germination not only increase the content of mung bean proteins and promote the hydrolysis of macromolecular subunits, but also facilitates the formation of small molecule subunits or polypeptides.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Comparison Study of Differential Expression Genes and cis-NATs of Skeletal Muscle Between Lantang and Landrace Pig

XU YueYuan, QI XiaoLong, HOU Ye, ZHAO YunXia, LUAN Yu, ZHOU HuanHuan, ZHAO ShuHong, LI XinYun

Scientia Agricultura Sinica. 2018, 51(9):  1795-1805.  doi:10.3864/j.issn.0578-1752.2018.09.016

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【Objective】 This study was designed to analyze the molecular mechanism of pig skeletal muscle development regulated by cis-natural antisense transcripts (cis-NATs) by comparing the differential expression genes and cis-NATs between Lantang and Landrace pig during their skeletal muscle development. 【Method】In this study, the following steps were adopted: (i) The differential expression genes (DEGs) and cis-NATs comparing Lantang (LT) and Landrace (LR) pig breeds at 10 time-points distributed from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) were identified (|log₂FC|＞1 and FDR＜0.01). (ii) The significant biological process Gene Ontology (GO) terms (P＜0.01) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (P＜0.05) of the DEGs and the sense gene of differential expression cis-NATs were enriched via function enrichment analysis. (iii) Then the significant biological process GO terms and KEGG pathways that cis-NATS participated were filtered by the number of related genes of cis-NATs, and KEGG pathways were integrated by the number of same genes between pathways. Finally, visual analyses was conducted based on fold change of differential expression cis-NATs and their sense gene between breeds within pathways.【Result】A total of 5 350 DEGs and 738 differential expression cis-NATs were identified between LT and LR at 10 time points in this study. GO analysis showed that the expression of differential expression cis-NATs between LT and LR in skeletal muscle was primarily concerned with genes in biological process, including muscle development and energy metabolism. The integrated analysis of KEGG pathways found that energy metabolism pathways were strongly related by DE genes and their correlated cis-NATs. Moreover the expression of genes and their correlated cis-NATs were higher in pig mitochondrial citrate cycle pathway during early stages of birth than adulthood. The visual analyses of KEGG pathways showed that the expression level of energy metabolism pathway involved genes and their correlated cis-NATs were higher in LT than LR at 49 dpc and 77 dpc, which were two critical points for muscle fibers growth. The differences of expression patterns between LT and LR primarily appear at the early postnatal stages of 2 dpn-90 dpn. 【Conclusion】In this study, 738 differential expression cis-NATs were identified by comparing between LT and LR at 10 time points of skeletal muscle development. Function enrichment analysis showed that cis-NATs played a  role in developmental differences of skeletal muscle fibers between LT and LR by interacting with genes expressed in energy metabolism related pathways.

Quantitative Determination of Diludine in Animal Feeds by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

XIAO ZhiMing, WANG Jun, SUO DeCheng, WEI ShuLin, JIA Zheng, LIU ChengXin, FAN Xia

Scientia Agricultura Sinica. 2018, 51(9):  1806-1814.  doi:10.3864/j.issn.0578-1752.2018.09.017

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【Objective】As a new kind of feed additive, diludine has been widely used in livestock production because it can promote the growth of animals, increase efficiency of the feed, and improve the meat quality. However, diludine is not allowed to use as a medical feed additive. The residues of diludine in animal origin foods may cause severe hypotensive reaction in the sensitive population. In the past World Consumer Rights Day, also known as 3.15, the national broadcaster China Central Television (CCTV) exposed parts of illegal use of veterinary drugs (e.g. diludine) in some of the feed companies, which draw extensive concerns of the society and the government. It is, therefore, of great importance to develop sensitive and reliable analytical methods to monitor diludine in feedstuffs in order to protect consumer rights and to provide technical support for government regulation.【Method】Chromatographic separation was achieved using a BEH C₁₈ column, and different mobile phases consisting of methanol-water, acetonitrile-water, methanol-0.1% formic acid, and acetonitrile-0.1% formic acid at different concentrations were optimized. In order to achieve the maximum sensitivity of ESI-MS/MS, direct infusion of standard solution was carried out in positive ionization mode to optimize the ESI source parameters (e.g. capillary voltage, cone voltage, and desolvation gas flow rate). Then dissociation with argon was induced and different collision energies and dwell times were compared in order to find the daughter ions, and the most abundant product ion was used as the quantification ion. In order to find the best sample preparation method, different extraction solvent (methanol and acetonitrile) and solid phase extraction (SPE) cartridges (C₁₈, HLB, MCX and alumina B) were optimized. 【Result】The optimized sample preparation procedures were conditioned as follows: Feed samples were extracted using acetonitrile and the supernatants were evaporated to dryness under a gentle stream of nitrogen at 60℃. The residues were re-dissolved with acetonitrile/water (1:9), and then cleaned up on HLB SPE cartridges. The target compounds were identified and quantitatively determined by ultra-performance liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (UPLC-MS/MS) operated in multiple reaction monitoring mode (MRM). The molecular ion [M+H]+ of diludine is m/z 254, and the loss of –OCH₂CH₃ (46 Da) is found to be common and leads to the major daughter ion m/z 208. The elimination of –CO (28 Da) from the ion m/z 208 gives the fragment at m/z 180, and then a subsequent loss of –COOCH₂CH₃ (73 Da) leads to the fragment at m/z 108. Therefore, in this study, the quantitative ion was m/z 254＞208, while m/z 254＞180 was used for qualitative ion. Under the current optimized chromatographic conditions, each LC run was completed in 10 min. Good linearity was obtained in the ranges of 0-500 μg·L^-1, with linear coefficients (R²) higher than 0.9992. The limits of detection (LODs) and quantification (LOQs) which defined as the concentration with a signal-to-noise ratio (S/N) of 3 and 10, were 10 μg·kg ^-1 and 50 μg·kg ^-1, respectively. Average recoveries from three fortification levels (10, 50 and 100 μg·kg ^-1) ranged between 82.6% and 101.0%, with relative standard deviations (RSD) lower than 9.2%. 【Conclusion】 The proposed method is fast, sensitive, and easy to perform, making it applicable for high-throughput daily monitoring.