Table of Content

16 October 2014, Volume 47 Issue 20

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES

Proteome Analysis of Cytoplasmic Male Sterility and Its Maintaince in JA-CMS Cotton

Scientia Agricultura Sinica. 2014, 47(20):  3929-3940.  doi:10.3864/j.issn.0578-1752.2014.20.001

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【Objective】This study was conducted to analyze and compare proteomes of cotton flower buds at the microspore abortion stage between a cytoplasmic male sterile (CMS) line JA-CMS and its maintainer line JB.【Method】Two-dimensional gel electrophoresis (2-DE) technique was used to separate the protein spots of JA-CMS and JB flower buds at the microspore abortion stage and the gel was stained with Coomassie Blue R-350 solution. Differentially expressed (DE) protein spots were selected with more than 2-fold changes and P values less than 0.05 by PDQuest (version 8.0.1) image software. LC-Chip/ESI-QTOF-MS analysis was carried out to obtain peptide mass fingerprinting of the DE protein spots. The Mascot software was used to search against the NCBInr database for protein annotation. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed to further explore the biological functions of the DE proteins.【Result】The 2-DE maps of JA-CMS at the sporogenous cells stage (SS) and microsporocyte stage (MS) isolated 1 525 and 1 540 protein spots, respectively, while 1 554 and 1 540 protein spots were detected in JB at the same stages. These protein spots were found within Mr 10-100 kD and pI 3-10. Fifteen DE protein spots between JA-CMS and JB were selected after the quantitative and statistical analysis. Among the 15 proteins, H(+)-transporting ATP synthase, glutathione reductase, ATPase subunit 1 (mitochondrion), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, and UDP-D-glucose dehydrogenase were differentially expressed at both the SS and MS stages between JA-CMS and JB. Anx1, S-formylglutathione hydrolase, momilactone A synthase-like, an unnamed protein product with allene oxide cyclase activity, and a conserved hypothetical protein located in mitochondria were down-regulated only at the SS stage in JA-CMS; beta-hydroxyacyl-ACP dehydratase, pyruvate dehydrogenase alpha subunit, and a predicted protein related with pollen development were differentially expressed only at the MS stage between the two materials. Functional category analysis indicated that these DE proteins were mainly involved in processes crucial for microspore and tapetum development. Down- and up-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in abnormal microspores and defective tapetum which leads to male sterility in JA-CMS ultimately. Quantitative RT-PCR was used to validate Rubisco and H(+)-transporting ATP synthase gene expressions at the transcriptional level, which are consistent with the protein amounts detected dy 2-DE.【Conclusion】CMS phenotype of JA-CMS may be associated with energy metabolism disturbance, abnormal jasmonic acid biosynthesis, accumulation of reactive oxygen species (ROS), and decreased activity of chalcone synthase (CHS). CMS is regulated by a complex signal network, multiple genes of different metabolic pathways may affect pollen fertility.

Association Mapping of Agronomic Traits in Soybean (Glycine max L. Merr.) and Mining of Novel Alleles

YANG Sheng-xian, NIU Yuan, LI Meng, WEI Shi-ping, LIU Xiao-fen, Lü Hai-yan, ZHANG Yuan-ming

Scientia Agricultura Sinica. 2014, 47(20):  3941-3952.  doi:10.3864/j.issn.0578-1752.2014.20.002

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【Objective】Mining stable quantitative trait loci (QTL), their novel alleles and the corresponding accessions for agronomic traits in soybean can provide important foundation for subsequent studies, such as molecular marker assisted selection and molecular breeding by design. 【Method】There were 135 simple-sequence repeat (SSR) markers uniformly distributed on the soybean genome which were used to scan 257 soybean cultivars obtained by stratified random sampling from six geographic ecotypes in China so that molecular marker information was gotten. Plant height, number of branches, number of nodes on main stem, stem diameter and number of pods per plant for each cultivar were measured at Jiangpu Experimental Station of Nanjing Agricultural University in 2009 and 2010 so that their phenotypic observations were obtained. The above phenotypic observations for each trait, along with the above molecular marker information, in the 257 soybean cultivars were used to carry out association analysis using generalized linear model (GLM), enriched compression mixed linear model (ECMLM) and epistatic association mapping (EAM) approaches. 【Result】Coefficients of variation for the above traits ranged from 24.62% to 52.34%, and significant differences at the 0.01 level among cultivars and for cultivar-by-environment interaction were observed, indicating abundant genetic variation in the mapping population. Forty-seven, eleven and fifty-eight commonly main-effect QTL across two years were detected by the above three approaches, respectively. Four (thirty-four) commonly main-effect QTL were identified across the above three (two) methods. One epistatic QTL and 32 QTL-by-environment interactions were identified by epistatic association mapping. Eighteen QTL identified in this study were consistent with (or linked to) those in previous studies. Some novel alleles for each agronomic trait and their corresponding accessions were mined. For example, satt669-145bp, satt102-154bp, satt382-395 bp and satt534-161bp were novel alleles for number of nodes on main stem, number of branches, stem diameter and plant height, respectively; and the corresponding accessions were Baihuadou, Hefeiliangtangjiaoshuangqingdou, Youhulu and Puxiadadou. 【Conclusion】One hundred and seven stable main-effect QTL for the above traits were detected across years or across methods.

QTL Mapping of 1000-Seed Weight in Brassica napus by Using the High Density SNP Genetic Map

JIAN Hong-ju, WEI Li-juan, LI Chao, TANG Zhang-lin, LI Jia-na, LIU Lie-zhao

Scientia Agricultura Sinica. 2014, 47(20):  3953-3961.  doi:10.3864/j.issn.0578-1752.2014.20.003

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【Objective】 Yield is the most complex trait in crops. It is directly determined by three yield-component traits (seed weight, pod number and seed number per pod). Increase of seed weight of rapeseed is the objective for breeding and genetic research. QTL mapping of seed weight in Brassica napus under five environments was conducted by using the high density SNP genetic map. which was constructed by the rape engineering technology research center of chongqing【Method】The reference SNP genetic map contains 2 795 SNP markers, covering 1 832.9 cM of B. napus genome, and an average distance of adjacent marker is 0.66 cM. QTL mapping of seed weight was conducted by composite interval mapping using software Windows QTL Cartographer. In order to find out the candidate genes with seed weight in B. napus, 49 genes associated with seed weight were collected in Arabidopsis thaliana and the homology region was searched using blastn in the QTL confidence interval with the E value<1E^–21. 【Result】 Larger ranges in five environments were detected, and the seed weight in five environments presented gaussian distribution, conforming the requirements of the QTL mapping. Trait analysis indicated that the seed weight among five environments was positively correlated at different levels. The seed weight in the environment of 2013 in Beibei showed a positive correlation with the seed weight in 2012 in Beibei and in 2008 in Giessen, and the correlation coefficients were 0.248 and 0.249, respectively. The seed weight in the environment of 2012 in Beibei showed a significant positive correlation with the seed weight in 2010 in Beibei, 2011 in Beibei and 2008 in Giessen, and the correlation coefficients were 0.226, 0.397 and 0.19, respectively. Fourteen QTLs located on 9 chromosomes were detected in five environments. And the LOD value ranged from 2.56 to 6.05. The QTL accounted for the phenotype variation from 4.64% to 14.13%. The genetic map and physical map comparison discovered that sixteen seed weight genes located on eight QTL’s confidence interval. Seven seed weight genes were detected in QTL qTSWA07-2, and the TTG2 gene was found in both qTSWA03-1 and qTSWC02-12. Interestingly, AHK3 was detected in three QTLs, namely qTSWA07-2, qTSWA08-1 and qTSWC01-1.【Conclusion】The seed weight QTL under five environments were mapped with a new set of rapeseed 60K chip array, and the mapping results are helpful to the seed weight QTL comparison in different materials based on the same rapeseed 60K chip array and the candidate gene analysis.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Alleviation of the Adverse Effects of Salt Stress by Regulating Photosynthetic System and Active Oxygen Metabolism in Maize Seedlings

ZHAO Ying, YANG Ke-jun, ZHAO Chang-jiang, LI Zuo-tong, WANG Yu-feng, FU Jian, GUO Liang, LI Wen-sheng

Scientia Agricultura Sinica. 2014, 47(20):  3962-3972.  doi:10.3864/j.issn.0578-1752.2014.20.004

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【Objective】This study aims at exploring and comparing the effects of exogenous monosaccharide glucose (Glc) and disaccharides sucrose (Suc) on growth, physiological and biochemical mechanisms in maize seedlings under neutral sodium salt stress.【Method】The university bred maize (Zea Mays L.) variety Kenyu No.6 was used in this study and was cultivated in 1/2 Hoagland nutrient solution in plant growth chamber. Seedlings at two-leaf stage were pretreated by 1/2 Hoagland nutrient solution containing 0.5 mmol·L^-1 Glc, Suc and mannitol for 3 days, seedlings left in normal nutrient solution and isotonic maninitol were used as control and permeability control, respectively. Then the plants were cultured in 1/2 Hoagland solution with NaCl concentration at 150 mmol·L^-1 NaCl for 4 days or 6 days. After 4 days of salt treatment, the full expanded 2rd leaves were used to exam physiological and biochemical indicators related to photosynthesis and antioxidant metabolism. After 6 days of salt stress treatment, maize samples were used for measuring plant height, root length and dry weight. The Li-6400XTR photosynthetic apparatus was applied for the determination of dynamic photosynthetic parameters on day 2, 4, and 6 of salt stress.【Result】Compared with the control, Glc and Suc pretreatment increased root length, dry matter and leaf relative water content, significantly increased plant height by 6.39% and 4.86%, respectively, and maintained higher net photosynthetic rate (P_n). There were no significant differences in other physiological and biochemical indicators between sugar pretreatment and control. Meanwhile, salt stress significantly inhibited the growth of maize seedlings. The height and root length of seedlings under salt treatment for 6 days decreased by 16.37% and 12.17%, and the aboveground and underground dry weight decreased by 20.69% and 25.00%. The antioxidative enzyme activities were decreased and the degree of membrane lipid peroxidation was increased under salt treatment. In addition, salt stress also significantly induced the decreases in P_n, stomatal conductance (G_s), transpiration rate (T_r), Hill reaction activity, intrinsic photochemical efficiency (F_v/F_m), photosynthetic pigment contents, and the intercellular CO₂ concentration (C_i) was firstly increased and then decreased with the extension of treatment time. However, exogenous Glc and Suc showed a significant reversal of salt stress on growth and accumulation of dry matter in maize seedlings. Plant height, root length, aboveground and underground dry weight of seedlings under Glc pretreatment were 6.12%, 4.49%, 15.65% and 4.49% higher than those under salt treatment, and those with Suc pretreatment increased by 7.34%, 4.87%, 17.39% and 7.34%, respectively. Glc and Suc pretreatments also alleviated the decreases of P_n, G_s, T_r and F_v/F_m, increased the Hill reactivity, relative water content and photosynthetic pigment contents due to salt stress. Moreover, the SOD, APX, and GPX activities in leaves of maize seedlings were increased markedly with sugar pretreatment, thus decreased the amount of thiobarbituric acid reactive substances (TBARS) and H₂O₂ contents. The effects of Suc pretreatment on removing H₂O₂ and improving Hill reactivity were better than Glc pretreatment. In addition, compared with salt treatment, mannitol pretreatment failed to induce any changes of the physiological and biochemical indicators, except to Hill reaction and chlorophyll a. In other words, mannitol pretreatment did not result in improvement of biological indicators of maize seedlings.【Conclusion】These results indicated that Glc and Sucpretreatments could promote the growth of aerial parts of maize seedlings by maintaining high P_n of leaves under normal environment. The sugar-induced salt stress resistance was associated with improved P_n in leaves, enhanced photosynthetic membrane system stability and photosynthetic pigment contents, activated the activity of PSII reaction, and improved the degree of stomatal opening, and carbon assimilation ability.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effect of Light Quality on Photosynthesis and Photosystem of  Maize (Zea mays L.) Leaves

ZHANG Shan-ping, FENG Hai-juan, MA Cun-jin, LI Geng, LIU Peng, DONG Shu-ting, ZHAO Bin, ZHANG Ji-wang, YANG Jin-sheng

Scientia Agricultura Sinica. 2014, 47(20):  3973-3981.  doi:10.3864/j.issn.0578-1752.2014.20.005

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【Objective】The effect of different light qualities on photosynthesis and photosystem of maize leaves was studied to explore the physiological mechanism of photosynthesis in cloudy.【Method】Two maize varieties were chosen as materials. Zhengdan 958 (ZD958) is a common variety mainly planted in northern China, and Xianyu 335 (XY335) is sweeping in the Northeast of China. The two varieties were planted in field at National Maize Technology Innovation Center of the Huanghuaihai Region. Four light quality treatments were designed, including red film (R), green film (G), blue film (B) and white gauze simulation on cloudy days as a control (CK) at the stage of V6. After 10 days of treatment, the parameters of gas exchange, chlorophyll a fluorescence transient and light absorbance at 820 nm of maize leaves were measured for analyzing the effects of different light qualities on photosynthesis and photosystem.【Result】The percentum of radiant energy of different light wave bands in overall radiant energy was the same with sunny days in cloudy days and simulated-cloudy days, but its absolute amount were significantly declined and the reduction of blue-violet was the largest. Blue film's decline was minimum at 300-510 nm in the three films. On the other hand, the percentum of radiant energy of blue film at 300-510 nm increased significantly than sunny days. The results showed that leaves net photosynthetic rate (P_n) of XY335 and ZD958 decreased significantly under green film, red film and blue film, and the decrease was 40.13%, 32.68%, 22.00% and 46.92%, 37.69%, 27.46%, respectively. Compared with the control, the stomatal conductance (G_s) was decreased significantly, but intercellular CO₂ concentration (C_i) was increased significantly, which demonstrated that the declines of P_n were not mainly related to stomatic factors under different light qualities. The probability of that a trapped exciton the moves an electron further than Q_A by trapped exciton (Ψ_o) and performance index (PI_ABS) of maize leaves were all declined significantly besides XY 335 under blue film, showing G＞R＞B. This demonstrated that the performance of PSⅡ was restrained significantly, especially the performance of the electron transport chain after PSⅡ reaction center electron acceptor side. The performances of electron donor side and acceptor side of PSⅡ were all declined significantly besides XY 335 under blue film. This demonstrated that there is a little effect of blue film on electron donor and acceptor. However, the decrease of activity of electron donor side was larger than the electron acceptor side under green film, but it was opposite under red film. It was found that the performance of PSⅡ (Ψ_o) was declined under different light qualities besides XY 335 under blue film. Most importantly,the performances of PSⅠ(ΔI/I_o) and Φ_{（PSⅠ/ PSⅡ）}of two varieties were all declined under different films, showing R＞G＞B.【Conclusion】The decline of blue-violet of visible light made the performance of PSⅠ dropped significantly in cloudy days, which resulted in the depressed coordination between PSⅡ and PSⅠ. And then the performance of electron transport chain in photosynthesis declined, and caused P_n decline.

PLANT PROTECTION

EST-SSR Information Analysis and Markers Development in Fusarium oxysporum

LI Xin-feng, ZHANG Zuo-gang, WANG Jian-ming, GAO Jun-ming, TIAN Hong-xian

Scientia Agricultura Sinica. 2014, 47(20):  3982-3991.  doi:10.3864/j.issn.0578-1752.2014.20.006

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【Objective】 The objective of this study is to investigate the distribution of Fusarium oxysporum SSRs (simple sequence repeat) derived from ESTs (expressed sequence tag) and to develop EST-SSR markers. These researches could provide technical supports for the genetic diversity analysis of Fusarium. 【Method】 All the 9 304 EST sequences of F. oxysporum were obtained from NCBI. SSRIT (simple sequence repeat identification tool) was used to search SSRs in these sequences and Primer Premier 5.0 was used to design primers. Fourteen pairs of primers were used to analyze the genetic diversity of 23 F. oxysporum strains from different hosts and areas, and the PCR products of these primers were detected by PAGE.【Result】A total of 92 SSRs were identified from 90 F. oxysporum EST sequences, with the frequency of 1.0%. The dinucleotide repeats (59.78%) were most abundant followed by tri-(17.39%) and penta-nucleotide repeats (11.96%). AC/GT (0.51%) occurred more often than any other repeat motif. Among the 30 synthesized primers, 20 (66.7%) amplified distinct bands, and 14 (46.7%) amplified expected polymorphic products. The genetic diversity analysis based on the SSR data showed that a total of 62 bands were amplified by 14 selected SSR primers, 59 (95.2%) of which were polymorphic, with an average of 4.2 bands per primer. The genetic similarity ranged from 0.487 to 0.933, with an average of 0.686. The genetic variation of the strains was obvious. The cluster analysis based on the SSR data showed that except No.14 strain, F. oxysporum strains were distinctly clustered into different groups according to their hosts at 0.781.【Conclusion】Exploitation of SSR markers from F. oxysporum ESTs is convenient and practicable. Fourteen EST-SSR markers identified in this study would be useful for genetic variation study of F. oxysporum.

Identification and Pathogenicity of Pathogen Casuing Anthracnose on Vaccinium

XU Cheng-nan, WANG Ya-nan, HU Tong-le, WANG Shu-tong, ZHOU Zong-shan, CAO Ke-qiang

Scientia Agricultura Sinica. 2014, 47(20):  3992-3998.  doi:10.3864/j.issn.0578-1752.2014.20.007

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【Objective】The objective of this study is to identify the pathogen causing stem and leaf anthracnose on Vaccinium in Liaoning, and to provide an IPM strategy for disease control and resistance breeding.【Method】Diseased samples were collected from Xingcheng and Zhuanghe County, Liaoning Province and disinfected stem and leaf pieces were placed on acidified potato dextrose agar (PDA) plate. Two represent isolates LNSW1 and B-Cg1 were chosen for morphological and molecular study. Isolates were characterized by colony morphology, conidial characteristics under 28℃ in dark. The internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) of isolates LNSW1 and B-Cg1 were amplified and sequenced. The consensus ITS sequences of isolates LNSW1 and B-Cg1 were compared with Colletotrichum spp. associated sequences in GenBank. Mega5.1 was used to draw the phylogenetic tree of isolates LNSW1, B-Cg1 and other related sequences. Another molecular identification of the two isolates were carried out with amplification of species-specific primer pair CaInt(CaInt2)/ITS4. Pathogenicity of both species was verified by mycelium plug inoculation on eight attached Vaccinium cultivars.【Result】Thirty-six ColletotrichumAB729126, KF698729, and EU886755 were divided into one group, and isolate B-Cg1 and CJF923828, KF516931, and GU174547 were divided into another group, the homology of each isolate in two groups was between 99%-100%. Additionally, the species-specific amplification results showed that both primer pairs could clearly distinguish the two different isolates. Pathogenicity tests results showed that the two Colletotrichum【Conclusion】Identification of the pathogen was based on morphology of colony and conidial, pathogenicity tests, nucleotide sequence of rDNA-ITS and species-specific primer pair CaInt(CaInt2)/ITS4. The typical anthracnose symptoms ofVaccinium in Liaoning Province were described in this study, disease diagnosis and pathogen identification was carried out based on the morphological characteristics, molecular technique and pathogenicity tests. Combined these results, it was confirmed that the causal agent of Vaccinium anthracnose in Liaoning were C. acutatum and C. gloeosporioides. spp. could both cause anthracnose lesions on leaves and stems of Vaccinium. The pathogenicity of C. acutatum was a little higher than C. gloeosporioides on Northern Highbush Vaccinium.. gloeosporioides isolates isolates were collected from Liaoning Province, two different groups were divided by morphology. Represent isolates LNSW1 and B-Cg1 were different in morphology of colony and conidia, and were skeptically identified as C. acutatum and C. gloeosporioides. The rDNA-ITS sequences length of isolates LNSW1 and B-Cg1 was 557 and 547 bp. The phylogenetic analyses showed that isolate LNSW1 and C. acutatum isolates

Effects of Fertilization on Aggregate Characteristics and Organic Carbon Distribution in a Paddy Soil in Dongting Lake Plain of China

WU Sheng-yong, WANG Peng-xin, ZHANG Zhi-ke, XU Xue-nong, LEI Zhong-ren

Scientia Agricultura Sinica. 2014, 47(20):  3999-4006.  doi:10.3864/j.issn.0578-1752.2014.20.008

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【Objective】 The objective of this study is to assess the capability of predatory mite carrying conidia and test the vitality of conidia, which will provide a theoretical basis for combining application of Beauveria bassiana with predatory mite against Frankliniella occidentalis. 【Method】 Neoseiulus barkerimL B. bassiana suspensions, the potential of conidia carried by predator was observed under scanning electron microscope. The number of conidia carried by predator at different times (2, 12, 24 and 48 h) was counted under hemocytometer, and the vitality of conidia was tested by colonies counting. Moreover, the infection rates of conidia dispersed by predator at different times above were tested by bioassay. 【Result】The treated predatory mites could carry conidia. After 2 h, one female adult predator carried 4.12×10⁴ conidia. The number of conidia carried was decreased gradually with time increasing, after 48 h, one predator carried 7.95×10³ conidia. The conidia dispersed by predators had vitality. After 2 h, the conidia dispersed by 10 predators formed 24 colonies on medium. The number of colonies was decreased gradually with time increasing, after 48 h, only 3 colonies could be formed. The conidia dispersed by predators were virulent to F. occidentalis. After 2 h, the conidia dispersed by 10 predators caused 75% mortality of adult F. occidentalis on the 10th day. The mortality of F. occidentalis was decreased gradually with time increasing, after 48 h, 25% mortality of adult F. occidentalis was caused. After 2, 12, 24 and 48 h, the mortalities of adult F. occidentalis over the 10 days displayed linear correlation. The lethal time (LT₅₀) after 2 h was 6.44 d, which was significantly lower than that after 48 h (9.83 d). 【Conclusion】Predator mites can carry a large number of conidia within a short time, the conidia have vitality and can be dispersed and infected F. occidentalis, therefore, combined application of B. bassiana and predatory mite have a potential for control of F. occidentalis. adults were sprayed by 1.0×10⁸ conidia/

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

Effects of Fertilization on Aggregate Characteristics and Organic Carbon Distribution in a Paddy Soil in Dongting Lake Plain of China

LI Wen-jun, YANG Ji-feng, PENG Bao-fa, CUI Jing-zhen

Scientia Agricultura Sinica. 2014, 47(20):  4007-4015.  doi:10.3864/j.issn.0578-1752.2014.20.009

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【Objective】As an important soil physical attribute, soil aggregates are the major site for soil organic carbon sequestration, so the impacts of long-term fertilization on aggregates characteristics and organic carbon distribution in a reddish paddy soil were studied in Dongting Lake Plain, China, which can provide a theoretical evidence for fertilization management in double cropping rice area. 【Method】Undisturbed soil samples at 0-20 cm layer were collected from a 27 years (1986-2013) field experiment, which was established in the Hanshou county in Hunan province. The experiment included 4 treatments: CK (without fertilization), NPK (N, P, K fertilizer application), LOM (combined application of NPK and 30% organic manure fertilizers), HOM(combined application of NPK and 60% organic manure fertilizers). All soils were separated into five aggregate-size classes (＞5 mm, 2-5 mm, 0.5-5 mm, 0.25-0.5 mm and ＜0.25 mm) by wet sieving method and the macro aggregates fractions (＞0.25 mm) were chosen for results analysis. The amount, mean weight diameter, geometric mean diameter, fractal dimension of water-stable aggregates which denoted by WR_0.25, MWD, GMD and D were exampled, separately. The aggregate-associated organic carbon was also measured.【Result】In addition to the size group of ＞5 mm, along with decreasing of particle sizes, the proportion of water-stable aggregates and the contribution rate of aggregates fractions to soil organic carbon (SOC) in the treatments increased, while the water-stable aggregate-associated organic carbon presented an opposite trend. Both the proportion of aggregates in the 2-5 mm and 0.5-2 mm size fractions were significantly increased under fertilization treatments when compared to CK treatment and the increasing rate was up to 35.5%-64.5% and 6.2%-14.7%, respectively. Fertilizer application also increased the value of WR_0.25, MWD, and GMD of aggregates by 8.6%-12.5%, 7.1%-15.1%, and 13.7%-28.4%, respectively. As for the fractal dimension, its value of fertilization treatments decreased by 2.3%-3.5% than that of CK, which suggesting that long-term addition of chemical N, P, K fertilizers or organic manure was beneficial to the improvement of soil structure. Long-term fertilization significantly increased the contents of organic carbon in various sizes of aggregates and the effect was much more obvious when combined application of organic manure and chemical N, P, K fertilizers, the content of aggregate-associate organic carbon of HOM treatment reached the highest, with the amplification up to 22.1%-36.6% when compared to CK. It also showed that 69.9%-83.5% of the SOC was concentrated in the macro water-stable aggregates and the percentage among different treatments decreased in the order of HOM＞LOM＞NPK＞CK. As for the contribution rates of aggregates to bulk SOC, there was a significant increase when the aggregates with size fractions ＞0.5 mm under fertilization treatments, while the allocation rates of SOC in the aggregates with size fractions of 2-5 mm and 0.5-2 mm were more sensitive to fertilization application. The results of correlation analysis revealed that there exists a significant positive correlation between the percentages of soil water-stable aggregates with the size of ＞0.5 mm and the content of SOC, indicating that newly enriched SOC mostly appeared in the size group of ＞0.5 mm. 【Conclusion】After long-term chemical N, P, K fertilizers or organic manure application, the contents of macro water-stable aggregates and organic carbon in reddish paddy soil increased obviously. Compared with single chemical N, P, K fertilizers application, chemical manure mixed with 60% organic manure application showed more obvious effects on the improvement of soil structure and the enhancement of soil carbon sequestration and was considered as the best fertilization mode for the tested paddy soil.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION

Effects of Combined Foliar Zn Application with N or P Under Different Water and Nitrogen Managements on Zn Nutritional  Quality of Winter Wheat

LI Hong-yun, WANG Shao-xia, LI Meng, TIAN Xiao-hong, ZHAO Ai-qing, GUO Chun-hui

Scientia Agricultura Sinica. 2014, 47(20):  4016-4026.  doi:10.3864/j.issn.0578-1752.2014.20.010

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【Objective】To alleviate the Zn deficiency problems in human population, a field experiment was conducted to investigate the effect of foliar Zn combined with N or P on grain Zn nutritional quality of wheat under different soil water managements and soil N application rates. 【Method】 The experiment was conducted in Guan-zhong Plain with a split-split plot design. The main plot treatments were three soil water management treatments: conventional planting, plastic film mulching (ridge mulching with film and furrow planting with winter wheat), and supplemental irrigation (40 mm irrigation during the over-wintering stage). And the sub-plot treatments were three N application treatments: 0, 120 and 240 kg N·hm^-2. The sub-sub-plot treatments were: CK (distilled water), foliar Zn (0.3% ZnSO₄·7H₂O), foliar Zn+N (0.3% ZnSO₄·7H₂O+1.7% CO(NH₂)₂), foliar Zn+P (0.3% ZnSO₄·7H₂O+0.2% KH₂PO₄). Grain samples were analyzed for Zn, P, and protein concentrations. 【Result】 The obtained results showed that compared to conventional planting, supplemental irrigation significantly increased grain Zn concentration, but film mulching significantly decreased grain Zn concentration in 2011-2012; Grain P/Zn molar ratio was significantly decreased by supplementary irrigation (6.8%) in 2011-2012. Compared to N0 treatment, grain Zn concentration was significantly increased by the N120 and N240 treatments in 2010-2011, with an average of 2.3% and 7.4%, respectively. Besides, N120 and N240 significantly decreased grain P/Zn molar ratio, and the decrement was higher in N240 than in N120, which resulted in a significantly decreased grain P/Zn molar ratio by 33.0% in 2010-2011 and 25.5% in 2011-2012 when compared to the N0 treatment. Furthermore, N fertilizer application resulted in a significant increase in grain protein concentration, but a significant reduction in grain P concentration. Compared with the CK treatment, foliar Zn, foliar Zn+N, and foliar Zn+P treatments significantly increased grain Zn concentrations from 20.6 mg·kg^-1 to 40.3, 39.6 and 33.7 mg·kg^-1, with an average extent of 95.5%, 96.0%, and 63.5%, respectively. These results suggest that combined foliar application of Zn with N or P fertilizers are the efficient agronomic approaches to increase grain Zn concentrations. Meanwhile, the increase in grain Zn concentration in the foliar Zn+N treatment was 5.9 mg·kg^-1 higher than in the foliar Zn+P treatment. Foliar Zn, foliar Zn+N, and foliar Zn+P significantly reduced P/Zn molar ratio by an average of 48.0%, 47.5% and 38.5%, respectively. Foliar Zn application significantly decreased P/Zn molar ratio by 66.7% in 240 kg N·hm^-2 and supplemental irrigation treatment. Grain protein concentration was significantly enhanced by foliar Zn+N treatment, with an average of 5.4% in 2010-2011 and 11.2% in 2011-2012. The foliar fertilizer application induced grain Zn accumulation was maximized by N fertilizer application and supplemental irrigation. When compared to the control treatment (foliar spraying distilled water, 0 kg N·hm^-2, and conventional planting), the foliar Zn+N with 120 kg N·hm^-2 and supplemental irrigation treatment caused the highest increase in grain Zn concentration (110.1%), and the largest decrease in grain P/Zn molar ratio (64.5%). 【Conclusion】Optimal water management practice (supplemental irrigation) and N fertilizer application promoted grain Zn accumulation and increased grain Zn bioavailability in the potentially Zn deficient soils, but there was highly significant increase in grain Zn concentration and bioavailability in the foliar Zn combined with N or P application treatments. And the increase in grain Zn nutritional quality was higher when foliar Zn combined with N than foliar Zn combined with P. Based on the economic benefit and Zn nutritional quality, timely irrigation, moderate N application and foliar Zn combined with N will be an effective method to improve grain Zn nutritional quality of wheat grain, which has a bright promoting prospect in the regions with potentially Zn-deficient or Zn deficient soils.

Effects of Combined Foliar Zn Application with N or P Under Different Water and Nitrogen Managements on Zn Nutritional  Quality of Winter Wheat

YAN Jian-mei, HE Bing-hui, TIAN Tai-qiang

Scientia Agricultura Sinica. 2014, 47(20):  4027-4035.  doi:10.3864/j.issn.0578-1752.2014.20.011

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【Objective】The characteristics of soil erosion and nitrogen losses caused by rainfall under different fertilizer application levels and tillage methods in the cropping of winter wheat-summer maize in purple soil area in Chongqing were studied in order to provide a theoretical evidence for the agricultural production and coordinate land management to improve the ecological environment.【Method】Four kinds of fertilizer levels (treatments of CK, combined application of manure and fertilizer, chemical fertilization, chemical fertilizer with increasing fertilization) and two kinds of tillage methods (chemical fertilization, contour plowing with chemical fertilizer) were designed in an experiment in runoff plots for a long term (2008-2012) observation to study the effect of soil erosion and nutrient loss. 【Result】There was a significant influence of runoff, sediment and nutrient loss on different fertilizer application levels and tillage methods, the runoff, sediment and nitrogen loss in the control group (CK) were the largest, contour plowing and fertilizer reduced the total volume of runoff, sediment and nutrient. The loss amount of the runoff, sediment and total nitrogen in contour plowing decreased by 25.9%, 20.5% and 33.4%, respectively, compared with the loss amount of the runoff, sediment and total nitrogen in cross-plowing plots. The loss amount of the runoff, sediment and total nitrogen in combined application of manure and fertilizer decreased by 35.7%, 40.5% and 41.2%, respectively, compared with that in chemical fertilization in CK. The runoff and the sediment showed a power function relationship (y=ax^b, a＞0, b＞0). The nutrient loss and the runoff showed a linear relation (y=ax+b, a＞0, b＞0), but no significant relation with precipitation. In dry land, Nitrogen loss was given priority to with total digestible nutrients (TDN), that accounted for the proportion of total nitrogen was 62.1%, combined application of manure and fertilizer can reduced the proportion of total digestible nutrients (TDN) and NH₄⁺-N to total N.【Conclusion】In the purple soil, longitudinal ridges should not be adopted and fertilizer should be used. Contour plowing with chemical fertilizer and combined application of manure and fertilizer was an effective means to prevention and cure of soil erosion and runoff in Chongqing purple slope, the nitrogen lost by total digestible nutrients (TDN) with the runoff.

HORTICULTURE

QTL Mapping and Epistatic QTL for Clubroot Resistance Using a Chinese Cabbage×Turnip F₂ Population

LI Peng-peng, LIANG Shan, CHEN Bing, YU Sha, ZHANG Chun-yu, SI Long-ting, PIAO Zhong-yun

Scientia Agricultura Sinica. 2014, 47(20):  4036-4044.  doi:10.3864/j.issn.0578-1752.2014.20.012

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【Objective】 This study was focused on the identification of quantitative trait loci (QTL) and epistatic interaction for clubroot resistance in Brassica rapa. 【Method】 A Chinese cabbage inbred line ‘BJN’ (B. rapa ssp. pekinensis) as a maternal parent was cross-pollinated with a turnip inbred line ‘Siloga’ (B. rapa ssp. rapifera) to obtain F₁ seeds. An F₂ population consisted of 140 individuals from one bud self-pollinated F₁ plant was employed to construct genetic map. Ninety five F₂ individuals and their F₃ families were used for QTL mapping and epistatic analysis for clubroot resistance (CR). In addition to 22 markers linked to 7 previously mapped CR genes, 1214 public molecular markers developed in B. rapa were scanned for polymorphism between two parents. JoinMap 4.0 was employed to construct the genetic linkage map. Clubroot resistance test was carried out against the two parental lines and F_2:3 families with the field isolates of Plasmodiophora brassicae collected from Brassica napus milpa in Anhui’s Huangshan. The disease index (DI) was evaluated as the mean DI for each F₂ individual based on the results of F₃ seedlings. QTL detection was performed using the composite interval mapping function provided by Windows QTL Cartographer 2.5. QTL Network 2.0 was used to identify putative epistatic loci based on a mixed linear model. The association between the genotypes of CR QTL linked markers in an F₂ population and the mean DI value of each F₂ plant was analyzed with SPSS 18.0.0. Two-way analysis of variance (ANOVA) was used to study the interaction effects between CR QTL linked markers. The differences among the nine genotypic groups classified by two QTL linked markers (sau_um026 and BrID90197) were assessed by multiple comparisons with least significant difference test and Student-Newman-Keuls test. 【Result】 Clubroot resistance test showed that ‘Siloga’ was resistant to clubroot disease, while ‘BJN’ was susceptible. The skewed normal distribution of the disease index for clubroot disease in F_2:3 families indicated that clubroot resistance was controlled by a few major genes and multiple genes as quantitative traits. Among 266 polymorphic markers detected, 222 were mapped to 10 linkage groups with a total length of 1152.6 cM. The genetic map covered 88.6% of the Chinese cabbage reference genome, and contained 5 markers linked to 3 different CR genes (CRb, Crr1 and Crr2). Two QTLs for clubroot resistance were detected in ‘Siloga’. One major QTL (qPbBa3.1) was located on the chromosome A3, and accounted for 19.02% of the phenotypic variance. Another QTL (qPbBa8.1) with minor effect was distributed on A8, and explained 7.82% of the phenotypic variance. Based on the physical location of CR QTL linked markers on the reference genome, two CR genes CRa and CRb were found in the qPbBa3.1 region, qPbBa8.1 was adjacent to Crr1. In addition, one pair of additive × additive epistasis was found between qPbBa3.1 and qPbBa8.1 with a contribution rate of 6.58%. Two-way ANOVA analysis showed that there was a significant difference between sau_um026 and BrID90197 (P=7.22×10^-5), confirming the presence of epistatic effects between qPbBa3.1 and qPbBa8.1. One-way ANOVA analysis revealed a significant difference among the nine genotypic groups regarding to clubroot resistance (P=9.45×10^-10). Multiple comparisons showed that the groups with the homozygous genotype as the resistant parent ‘Siloga’ at the qPbBa3.1 locus were significantly different from other groups. The groups with heterozygous genotype were also significantly different from the groups that were homozygous as the susceptible parent ‘BJN’ at qPbBa3.1. The clubroot resistance could be improved by the presence of the qPbBa8.1 locus. 【Conclusion】 The clubroot resistance was controlled by a major qPbBa3.1 and a minor qPbBa8.1, and the epistatic interactions between them in ‘Siloga’.

Comparison Between Tetraploid Watermelon(Citrullus lanatus) and Its Diploid Progenitor of DNA Methylation Under NaCl Stress

ZHU Hong-ju, LIU Wen-ge, ZHAO Sheng-jie, LU Xu-qiang, HE Nan, DOU Jun-ling, GAO Lei

Scientia Agricultura Sinica. 2014, 47(20):  4045-4055.  doi:10.3864/j.issn.0578-1752.2014.20.013

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【Objective】 The morphological differences and DNA methylation changes of diploid and tetraploid watermelon seedlings after NaCl stress were studied, combined with morphological indicators, this experiment aimed at explaining the resistance mechanisms between diploid and tetraploid watermelon seedlings from the perspective of epigenetics.【Method】Three groups of different ploidy (diploid, tetraploid) watermelon seedlings were used in this experiment, the watermelon seedlings were treated by using 1/2 Hoagland nutrient solution with NaCl concentration at 0, 100, 200, 300, and 400 mmol^.L^-1 when the seedlings had three euphyllas, after 8 days of NaCl treatment, the salt stress index of watermelon seedlings, the changes of methylation levels and patterns of the watermelon seedlings were analyzed by methylation-sensitive amplified polymorphism (methylation sensitive amplified polymorphism, MSAP) technology.【Result】 According the salt-damage morphological index comparison, it was found that after NaCl treatment, with the increasing NaCl concentration, the extent of injury of three groups watermelon seedlings also increased. There was a difference in degree of injury among three watermelon varieties, but at the same NaCl concentration, the degree of injury in the diploid watermelon seedlings was greater than tetraploid which was in the same variety. From the methylation level perspective, the whole genomic DNA methylation rate, half rate of methylation, the total rate of methylation of watermelon seedlings decreased with the increase of NaCl concentration, at the same NaCl concentration level, the methylation rate of diploid watermelon seedlings was bigger than the tetraploid ones, the methylation level was positively correlated with the degree of injury of the watermelon seedlings. From the methylation patterns, the DNA demethylation ratio decreased and the hypermethylation rate increased after NaCl stress, at the same time, the magnitude of the changes in the tetraploid watermelon seedlings was greater than the diploid ones, the methylation patterns of watermelon seedlings were negatively correlated with their degree of injury.【Conclusion】 The genomic DNA methylation status of watermelon seedlings was changed after NaCl stress, and the changes showed a high linear correlation with the NaCl stress degree. Watermelon seedlings got more tolerance to NaCl stress by reducing methylation rate and the methylation ratio, and the tetraploid watermelon seedlings showed stronger resistance than the diploid ones through decreasing the levels of methylation and methylation ratio.

Flesh Texture Characteristic Analysis of Pear

WANG Yan-xia, WANG Xiao-man, GUAN Jun-feng

Scientia Agricultura Sinica. 2014, 47(20):  4056-4066.  doi:10.3864/j.issn.0578-1752.2014.20.014

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【Objective】The aim of this study is to compare fruit texture characters of different pear cultivars and to investigate the optimum evaluation parameters for texture of pear, and to provide proves for the study on internal quality of pear fruit. 【Method】The characters of flesh texture were analyzed by texture profile analysis (TPA) (including frangibility, hardness, springiness, resilience, cohesiveness, chewiness) and puncture test (PT) (including fracture force, fracture work, fracture displacement, bio-yield force, bio-yield work, bio-yield displacement) on 14 different pear cultivars harvested at maturity stage, and the relationships among different texture parameters were obtained by multivariant analyses.【Result】There were certain differences at flesh texture based on TPA and PT among different varieties of pear. The varieties ‘Butirra Rostata Morettini’, ‘Mansoo’ and ‘Nansui’ showed higher flesh hardness, frangibility and chewiness than ‘Yali’, ‘Housui’, ‘Meirensu’ and other cultivars, and the ‘Butirra Rostata Morettini’, ‘Mansoo’ and ‘Mantianhong’ had higher fracture force, fracture work, bio-yield force and bio-yield work than ‘Yali’, ‘Housui’, ‘Hwangkum’ and other cultivars. Relatively, the displacements of fracture and bio-yield had smaller differences among different cultivars. The marked close correlations were observed among flesh texture parameters, such as frangibility, hardness, resilience, cohesiveness, chewiness, fracture force, fracture work, bio-yield force and bio-yield work, and most of them were positively correlated, except springiness, fracture displacement and bio-yield displacement. The 14 cultivars of pear were able to classify into 3 different texture kinds using cluster analysis, which were soft (such as ‘Housui’, ‘Hwangkum’, ‘Huangguan’, etc), crisp (‘such as Mansoo’) and hard (‘Butirra Rostata Morettini’), respectively. The result of principal component analysis (PCA) showed that the cumulative contribution ratio of the first three principal components to the fruit texture reached 92.196%. The first principal component (PC) reflected chewiness of flesh, the second springiness and yield, and the third fracture. This suggested that hardness, fracture force and frangibility had primary influence on flesh texture, and springiness and fracture displacement second. 【Conclusion】There are certain differences in the flesh texture among different cultivars of pear. Significant positive correlations were observed between most of TPA and PT parameters of the flesh. The flesh hardness, fracture force and frangibility are more important parameters to influence on fruit flesh texture in pear.

STORAGE·FRESH-KEEPING·PROCESSING

Identification of Anthocyanins in Brassica campestris L. and Their Stability and Antioxidant Activity

KUANG Min-jie, QI Min-yu, HE Jing-ren, LI Shu-yi, LIU Gang, ZHU Zhen-zhou, CAI Hong-yan, FENG Jun

Scientia Agricultura Sinica. 2014, 47(20):  4067-4077.  doi:10.3864/j.issn.0578-1752.2014.20.015

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【Objective】 To identify the specific compositions of anthocyanins from pigment extracts of Brassica campestris L. (BPE) and to investigate their chromic stability and antioxidant activities in order to provide a scientific basis for the deeply processing of B. campestris L and further the broad application in food industry production.【Method】 The anthocyanins components of BPE were analyzed by HPLC-PDA-ESI-MS. The effects of pH and SO₂ on stability of the pigments were investigated and their antioxidant activities were evaluated by assaying scavenging free radicals and reducing power respectively.【Result】For the first time fifteen different anthocyanins were tentatively identified in BPE pigments, mainly consist of nine highly acylated cyanidin-glycosides (cyanidin 3-sophoroside-5-malonylglucoside, cyanidin 3-caffeoylsophoroside-5-malonylglucoside, cyanidin 3-coumarylsophoroside-5-glucoside, cyanidin 3-feruloylsophoroside-5-glucoside, cyanidin 3-coumarylsophoroside- 5-malonylglucoside, cyanidin 3-feruloylsophoroside-5-malonylglucoside, cyanidin 3-sinapylsophoroside-5-malonylglucoside, cyanidin 3-(6′-malonylglucoside- 2′-(6″-coumaryl-2″-hydroxy benzoyl-glucoside))-5-(6-malonylglucoside) and cyanidin 3-(6′-malonylglucoside-2′-(6″-feruloyl-2″- hydroxy benzoyl-glucoside))-5-(6-malonylglucoside)) and two non-acylated monoglycoside and diglycosides of cyanidin (cyanidin 3-glucoside and cyanidin 3-sophoroside-5-glucoside), as well as a few non-acylated monoglycoside and diglycosides of delphinidin (delphinidin 3- glucoside and delphinidin 3-glucoside-5-glucoside) and petunidin (petunidin 3-glucoside and petunidin 3-sophoroside-5-glucoside). Their pH stability was similar to simple anthocyanins (stable under acidic conditions and becoming unstable with the increase of pH), but the pigments were very stable to resist the SO₂ bleaching. The abilities of scavenging free radicalsand reducing power of BPE pigments were enhanced as the concentration increased. Within the same concentrations the abilities to scavenge ·OH of BPE was equivalent as ascorbic acid (P>0.05) while the reducing power was lower than ascorbic acid. The ability to scavenge DPPH radicals of BPE was higher than ascorbic acid.【Conclusion】The B. campestris L. is rich in highly acylated and glycosylated anthocyanidins (flake of purple crystal gloss) with high stability and strong antioxidant activity, therefore, it is a new resource worthy to be developed into functional ingredients and applied products with anthocyanin pigments.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Research Progress in Muscular Growth and Development of Long Noncoding RNAs

WEI Cai-hong, WU Ming-ming, LIU Rui-zao, ZHAO Fu-ping, ZHANG Li, DU Li-xin

Scientia Agricultura Sinica. 2014, 47(20):  4078-4085.  doi:10.3864/j.issn.0578-1752.2014.20.016

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It is well known that the growth traits are complex traits which are jointly or mutually affected by genetic and environmental factors. Although the genome-wide association analyses can be used to analyze all genes on the genome and screen out SNPs associated with certain traits, it is hardly and comprehensively to evaluat the exact role of one gene. One of the goals of breeders is to find the precise gene associated with growth and development, the researches on gene expression and regulation in the past were mainly through the regulation of gene transcription which are mediated by transcription factors, and the protein-coding genes were of the biggest concern, and so the RNA once thought to be transition between proteins and DNA , but the role of RNA in the process of life is far more than people imagine, especially long noncoding RNA (LncRNA) which was recently discovered. LncRNA played a key role in almost all important life events, such as cell proliferation and differentiation, ontogeny, signal transduction, stem cell maintenance and metabolism and so on. It also could regulate the expression of genes on the epigenetic level, the level of transcription and post-transcriptional level, etc., and closely related to the occurrence of many major human diseases. The long noncoding RNA is longer than 200 nt and does not show the potential of coding protein. LncRNA exerts its biological function through multiple mechanisms, which participates in X chromosome silencing, genomic imprinting, chromatin modification, transcriptional activation, transcriptional interference, nuclear transport and other important regulatory process. And LncRNA relates to epigenetic regulation, transcriptional regulation and post-transcriptional regulation. Through in-depth understanding of LncRNA function to adjust the depth LncRNA new molecular pathways regulating muscle development and differentiation through myogenic factor, a new perspective to examine and explain the molecular events mammalian myogenic, and to find new LncRNA molecules in muscle tissue related to growth and development, and to find the LncRNA molecular which are closely related to growth and development and biological function of the target gene in-depth study, and to clarify the molecular mechanisms of LncRNA regulation in muscle growth and development, and it’s the main contents of the genetic breeding researches in terms of muscle development. This article focused the research progress on LncRNA’ s role in mammalian development, cell growth, differentiation and proliferation.

Research on Key Factors Restricting Efficiency of Sperm-Mediated Gene Transfer

JIAO Ming-xia, MU Yan-shuang, BOU Gerelchimeg, ZHANG Lin-lin, KONG Qing-ran, FU Hai-peng, LIU Zhong-hua

Scientia Agricultura Sinica. 2014, 47(20):  4086-4095.  doi:10.3864/j.issn.0578-1752.2014.20.017

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【Objective】 Sperm-mediated gene transfer technique is simple, it has been recognized by many scientists as a way to produce transgenic animals, however, the results from different laboratories showed poor stability and reproducibility, transgenic efficiency is also significantly different. The aim of this study is to clarify the main factors causing this phenomenon.【Method】Capacitated sperms were incubated with 0, 15, 30 and 300 nmol·L^-1 Cy-3 labeled DNA (Cy-3-DNA）at 37℃ for 30 min. After incubation, sperms viabilities were detected by CBC board. Sperms incubated by DNA were used to direct smeared, smeared after washing by DPBS and smeared after digested by Dnase I, sperm smears were placed under fluorescent microscope. The total number of sperms and the number of sperms which show Cy-3 signal in the vision were recorded, the records were used for the efficiency statistics of sperms binding and uptaking exogenous DNA. 50µmol·L^-1 P4 were used for inducing sperms acrosome reaction which were incubated with 300 nmol·L^-1 Cy-3-DNA. The proportion of sperms showing Cy-3 signals were checked before and after the acrosome reaction. Sperms incubated with 15 and 300 nmol·L^-1 Cy-3-DNA were used for IVF, the presence of Cy-3-DNA in the zygotes was detected under fluorescence microscopy. According to the optimized conditions for these experiments, sperms incubated with 0, 15 and 300 nmol·L^-1 pEGFP-C1 were used for getting transgenic embryos, and oocyte fertilization rate and embryonic development rate in experimental group and control group were compared, PCR and RT-PCR were used to detect the presence and expression of pEGFP in the blastocyst.【Result】Sperms after capacitation were incubated with 0, 30, 15, 30 and 300 nmol·L^-1 Cy-3-DNA, the sperm viability was 82.21%, 73.63%, 77.38%, 76.33% and 77.80%, respectively. The rate of positive sperms was 76%, 94%, 99% and 100% before washing , and that in 15，30 and 300 nmol·L^-1 treatment groups had more efficiency than 3 nmol·L^-1 (P＜0.05) group. The rate of positive sperms was 45%, 66%, 84%, 87% after washing and 44%, 56%, 71%, 76% after digestion, and that in 300 and 30 nmol·L^-1 treatment groups had more efficiency than the other two groups, and 15 nmol·L^-1 treatment group is had more effiency than 3 nmol·L^-1 (P＜0.05) group. The result showed that the amount of uptaking DNA increased significantly as the increasing of DNA concentration by ImageJ analysis(P＜0.01). After acrosome reaction, DNA attached on the acrosome would be lost, but DNA exist in the rear of the sperm head still retained, so the ratio of positive sperms did not decrease. Sperms incubated with 15 and 300 nmol·L^-1 DNA were used for IVF. There were exogenous DNA fluorescence signals distributed in zygotic embryos under fluorescence microscopy, embryos which fluorescence signals densely distributed in male pronuclear were recorded and considered as positive embryos. The positive rate of zygotic embryos derived from sperm incubated with 300 nmol·L^-1 DNA was 27.89%, which was significantly higher than that incubated with 15 nmol·L^-1 (P＜0.05). Sperms were incubated with pEGFP-C1 plasmid were used for IVF. The result showed that incubation did not affect the oocyte fertilization rate，and the embryonic development rate compared with the control group. Single blastocyst PCR detection showed that the transgenic blastocyst rate derived from sperms incubated with 15 and 300 nmol·L^-1 pEGFP-C1 was 8.72% and 21.50%, respectively. RT-PCR detection of EGFP expression in the blastocysts derived from 300 nmol·L^-1 DNA showed positive results but no protein expression was detected by fluorescence microscopy.【Conclusion】Sperms have the ability of binding and uptaking exogenous DNA, and sperms can carry exogenous DNA into oocytes. Acrosome reaction causes part loss of the DNA, but in the rear zone of the sperm head, DNA is still retained. The key factors restricting the efficiency of sperm-mediated gene transfer might be integration of exogenous DNA into the genome and expression activation of the exogenous genes during fertilization.

ANIMAL SCIENCE·VETERINARY SCIENCERE

Detection and Analysis of Resistance Genes in Quinolone-Resistant Escherichia coli Isolates from Different Livestocks in Xinjiang

NAN Hai-chen, DI Li-na, XIA Li-ning

Scientia Agricultura Sinica. 2014, 47(20):  4096-4108.  doi:10.3864/j.issn.0578-1752.2014.20.018

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【Objective】The objective of this study is to investigate the prevalence and characteristics of plasmid mediated quinolone resistance (PMQR) determinents in quinolone-resistant E. coli from different animal origins in Xinjiang, and their coexistence with the major β-lactamases and 16s rRNA methylation enzyme genes. 【Method】 Polymerase chain reaction (PCR) was used to detect PMQR (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB, aac(6’)-Ib-cr), b-lactamase gene (bla_TEM, bla_CTX-M, bla_SHV, bla_KPC, bla_CMY-2, bla_LAP-1) and 16S rRNA (armA, rmtB) genes in 79 strains from pigs, 8 strains from cattle and 96 strains from sheep quinolone-resistant (ciprofloxacin, norfloxacin, enrofloxacin) E. coli. The positive strains were performed by using DNA sequencing to determine the purpose of the belt.Minimal inhibitory concentrations (MICs) of the related antibiotics to these isolates carrying the β-lactamase and 16S rRNA methylase genes,were tested and the relationship between the genotype and resistant phenotype was analyzed.【Result】The results showed that the qnrS (6.33%, 5/79), aac(6')-Ib-cr (5.06%, 4/79), oqxA (44.3%, 35/79), oqxB (50.6%, 40/79) were main PMQR determinants, bla_TEM (100%, 79/79) was main β-lactamase genes and rmtB (3.80%, 3/79) was main16S rRNA methylase genes in E. coli from pigs; the qnrS (12.50%, 1/8), oqxA (12.5%, 1/8), oqxB (12.5%,1/8), aac(6')-Ib-cr (12.50%, 1/8) and qepA (12.50%, 1/8) were main PMQR determinants, the bla_TEM (100%, 8/8) and bla_SHV (12.50%, 1/8) were main β-lactamase genes in E. coli from cattle; the qnrS (6.25%, 6/96), oqxA (33.3%, 32/96) oqxB (38.5%, 37/96) and aac(6')-Ib-cr (22.91%, 22/96) were main PMQR determinants, the bla_TEM (100%, 96/96) and bla_CTX-M (2.08%, 2/96) were main β-lactamase genes and the rmtB (2.08%, 2/96) was main16S rRNA methylase genes in E. coli from sheep. The qnrA, qnrB, qnrC, qnrD, bla_KPC, bla_CMY-2 and bla_LAP-1 genes were not detected in any of the isolates. The co-harboring of resistant genes was common among these E.coli from different animals, and the resistant phenotype of E.coli from different animal sources carrying β-lactam enzyme and 16S rRNA methylase genes has some correlation.【Conclusion】The PMQR determinants were existed in E. coli from different animals in Xinjiang, and they coexist with the main b-lactamase and/or 16s rRNA methylation enzyme genes. In addition, the PMQR determinants and β-lactamase genes were firstly detected in the E. coli from sheep.

AGRICULTURAL ECONOMY & MANAGMENT

Study on Lawmaking About Fertilizer Use and Management in China

PENG Shi-qi

Scientia Agricultura Sinica. 2014, 47(20):  4109-4116.  doi:10.3864/j.issn.0578-1752.2014.20.019

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Fertilizer is the most important means of production in agricultural production. But the excessive fertilization and fake fertilizer on market bring the problems of the reduction of agriculture output, quality safety of agricultural productions, environmental pollution and even personal safety occasionally happened in China. Fertilizer legislation has become a calling under the wide public concern. In order to protect the environment and farmers' interests, many countries have fertilizer laws or related regulations with contents of the fertilizer use and management. They contain with fertilization according to the result of soil test, fertilizer nutrient standards, the fertilizers limited index in the nitrate vulnerable area, use time and quantity standards of organic fertilizer in the water resources protection area, farmers are in duty bound to record the fertilizer use and so on. The users must manage their fertilize use in accordance with the law. China has not a unified supervision and management system in fertilizer management. The fertilizer using rules and standards in agriculture production are by administrative management and project promotion. Absence of fertilizer use management has the serious influence to the sustainable utilization of agricultural resources and ecological environment protection and agricultural production. Fertilizer lawmaking including fertilizer use management has become the urgent needs. Some relevant laws in China have stipulated the responsibilities for the administrative departments in fertilizer use management. But more terms are guidance provisions without clear department’s duties, fertilizer use standard and limit, compulsory means and the enforced measures to fertilizer user. The legislation in fertilizer use management regulation should grasp three key points: (1) through product admittance system control fertilizer varieties and quality; (2) through special standards and environmental requirements control the quantity and period of the fertilizer use. (3) clear subject liability and establishing responsibilities and rights of all sides. This paper made a proposal concerning the legislation contents in fertilizer use and management including six aspects: (1) making clear access and exit mechanism about fertilizer products in agriculture use; (2) making clear the responsibilities and rights of the management body; (3) making clear and definiting the responsibilities and rights of the enterprises, dealers, the users; (4) stipulating the behaviors of the service organizations and individuals in providing technical service activities; (5) making clear and defining the security system and mechanism of management; (6) making clear and defining fertilizer quality standards, the use limit, use time and other key standards. Through fertilizer use and management legislation, fertilizer producers, agencies and users must comply with the regulation. It will promote fertilizer use management from administrative management to follow the laws and regulations. Based on the fertilizer use management rules, fertilizer production rules, sales behavior rules and punishment rules should be made more clearly so as to constitute a complete fertilizer law.

Study on Construction of Agricultural Informatization Taking Shandong Province as a Case

RUAN Huai-jun, FENG Wen-jie, TANG Yan, ZHAO Jia, LI Dao-liang

Scientia Agricultura Sinica. 2014, 47(20):  4117-4127.  doi:10.3864/j.issn.0578-1752.2014.20.020

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【Objective】 Informatization is the general developing trend in modern world, and also the important force to promote the economic and social reform of China. As one of the biggest agricultural provinces in China, the development of Shandong agriculture is increasingly constrained by resources, so it is urgent to upgrade and transform the agricultural industry. As the first demonstration province for rural and agricultural informatization in China, Shandong Province has made a prior attempt on promoting agricultural modernization by informatization. In this paper, the practice and effect of agricultural informatization development in Shandong were analyzed and summarized. Based on these, the huge effect of informatization on modern agriculture was further verified, and the referential empirical modes were summarized in order to provide references for the other provinces. 【Method】 The methods of field visit and panel discussions were used to know about the current situation and existing problems in the development of agricultural informatization in Shandong, and the quantitative research was carried out based on the data obtained from investigation. So the practice and effect of agricultural informatization construction of Shandong were mastered in a view of macro-perspective, the pointed policy measures were also put forward for the agricultural informatization. 【Results】 The construction of comprehensive provincial service platform for rural and agricultural informatization, integration of Three Networks information service high speed channel, grassroots information service system and demonstration project were conducted in Shandong Province, and also the exploration of long-term mechanism. At present, the comprehensive service platform has been preliminarily constructed and commissioned, and the constructed high speed channel is convenient with wide coverage; there were more than 2000 grassroots service stations were constructed within the whole province to carry out scientific information service, e-commerce and agricultural internet of things demonstration and so on in vegetables, fruit trees, domestic animals and domestic fowls, aquatic products, and other important industries. A plenty of informatization achievements have been popularized and applied, and the long-term mechanism has been constructed, thus, the modern agricultural development, new village construction and the integrative development of informatization with modernization of agriculture were promoted. 【Conclusion】 The agricultural informatization is a key support for the modern agricultural development, a significant embodiment of agricultural modernization level, an important content of agricultural modernization construction and also the key action to enhance the agricultural efficiency, increase farmers’ income and develop rural economy. It is the optimal path to develop agricultural informatization and realize Four-Modernization-Synchronization in Shandong Province even the whole nation through informatization to promote agricultural modernization.

RESEARCH NOTES

Impact of Biochar Application on Soil Nutrients and Microbial Diversities in Continuous Cultivated Cotton Fields in Xinjiang

GU Mei-ying, LIU Hong-liang, LI Zhi-qiang, LIU Xiao-wei, TANG Guang-mu, XU Wan-li

Scientia Agricultura Sinica. 2014, 47(20):  4128-4138.  doi:10.3864/j.issn.0578-1752.2014.20.021

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【Objective】 The objective of this experiment is to explore the effects of biochar application treatment on soil nutrients and microbial diversities in continuous cultivated cotton fields in Xinjiang. The results might provide a scientific basis and practical guidance for optimal utilization of agricultural wastes and prevention of the continuous cultivation problems in cotton fields.【Method】Under the field cultivation conditions, nutrient contents were determined，and traditional microbial cultivation method and Biolog microplate technique were used to study soil microbial communities, the number of physiological bacterial groups and microbial carbon utilization in rhizosphere and non-rhizosphere. Soil nutrients, microbial community characteristics and functional diversities of different biochar treatments were analyzed in grey desert soil and aeolian sandy soil in Shihezi irrigation zone. The experiment designs in grey desert soil were, respectively, biochar+conventional fertilizer (BC+CK) and CK treatments, and biochar application amounts were 22.5 t·hm^-2. The experiment designs in aeolian sandy soil were BC1+CK, BC2+CK and CK treatments, biochar application amounts of 22.5 (BC1) and 45.0 (BC2) t·hm^-2, respectively. 【Result】 Biochar application had influence on pH and nutrients in rhizosphere and non-rhizosphere soils in continuous cultivated cotton fields. Compared with CK, pH of grey desert soils decreased or had no significant difference, that in aeolian sandy soilsincreasedsignificantly. OM of two groups ofgray desert rhizosphere soilsincreased by 36.1% and7.9%, that in non-rhizosphere soilsincreased by 32.8% and15.4%, respectively. OM of aeolian sandy rhizosphere soilswith low and high biochar application increased by 63.6% and295.1%, that in non-rhizosphere soilsincreased by 93.5% and108.8%, respectively.There was no obvious regularity in nutrient contents ofgrey desert soils, available P and available K increased, but available N decreased in aeolian sandy soils. Biochar application increased, the numbers bacteria and fungi in rhizosphere soils in continuous cultivated cotton fields, and the effect in aeolian sandy soil was better than that in grey desert soil. The numbers of soil bacteria in rhizosphere of grey desert soils with biochar application rates increased by 2.2% and 72.1%, respectively, and the numbers of soil fungi increased by 80.0% and 83.3%, respectively. In contrast, the numbers of soil bacteria in rhizosphere in aeolian sandy soils with low and high biochar application rates increased by 16.1% and 35.7%, respectively, and the numbers of soil fungi increased by 300.0% and 300.0%, respectively. Furthermore, the amounts of Cytophaga and Azotobacter were enhanced by biochar application in rhizosphereand non-rhizosphere of grey desert soil, while the amounts of Nitrifier showed a decreasing trend. The amounts of physiological soil bacterial groups were significantly improved in rhizosphereand non-rhizosphere of aeolian sandy soil. Carbon source utilization of soil microbial community showed that soil microbial activities for treatment with biochar application had no significant difference or significantly improved, comparing with the control treatment. However, the effect on soil microbes in rhizosphere of aeolian sandy soil was better than that in non-rhizosphere soil, since theShannon richness index of rhizosphere soilhad a rising trend. 【Conclusion】 Biochar application could improve soil nutrients and microbial diversities in rhizosphere of continuous cultivated cotton fields in Xinjiang, while the soil improvement effect on aeolian sandy soil was better than that on grey desert soil.