Scientia Agricultura Sinica

What We Have Learnt in Ten Years′ Study of Rice Transgene Flow

JIA Shi-Rong-1, YUAN Qian-Hua-2, WANG Feng-3, YAO Ke-Min-4, PEI Xin-Wu-1, HU Ning-4, WANG Zhi-Xing-1, WANG Xu-Jing-1, LIU Wu-Ge-3, QIAN Qian-5

Scientia Agricultura Sinica. 2014, 47(1): 1-10. doi:10.3864/j.issn.0578-1752.2014.01.001

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China is the largest rice producer worldwide and is one of the origins of Asian cultivated rice as well. Along with the rapid development of transgenic rice in China, the potential impact of rice transgene flow on the environment and food safety has become one of the major concerns. Gene flow is an important parameter in the risk assessment and regulation of transgenic rice on the scientific basis. In accordance with this situation, we have formed a team and systematically studied the rice transgene flow since 2002. The results obtained in recent ten years are as following: (1) the patterns of transgene flow and the major biological and meteorological factors controlling rice gene flow have been elucidated. Following the prevailing wind direction in rice flowering period, a rectangular design of field experiments were conducted at 3 locations（Sanya, Hainan Island; Guangzhou, Guangdong; and Hangzhou, Zhejiang）in 2-3 years by using a homozygous transgenic line L201 or B2 (sister lines) with bar gene inserted, resistant to herbicide Basta, as a pollen donor, and totally 19 non-transgenic rice as recipients, including male sterile (ms) lines, common rice cultivars (CRC), F1 hybrid rice, and common wild rice (Oryza rufipogon). Results indicated that the frequency of transgene flow to ms lines was the highest, while gene flow to CRC and F1 hybrids was the lowest (less than 1% or 0.1% at parallel plantation). The frequency of transgene flow to O. rufipogon was in between. By comparison, the maximum frequency of gene flow to ms lines is one to three orders of magnitude higher than that to O. rufipogon and CRC. Gene flow frequency decreased exponentially as the distance increase, with a sharp cut-off point at about 1-2 m in Guangzhou and Hangzhou, while it was approximately 5m in Sanya. It indicates that the sharp cut-off point is closely related to the wind speed during rice flowering period at a given location. By using a concentric circle design of field experiment and an ms line BoA with higher outcrossing rate as a recipient, we have been able to clearly quantify the relationship between the gene flow frequency and the wind direction. On the basis of the cumulative data in eight compass sectors, 90%–96% of the cumulative gene flow events occurred in the four downstream prevailing wind sectors, while it was only 4%–10% in the four lateral and upstream prevailing wind sectors. In short, a general conclusion is that the order of magnitude of transgene flow frequency is basically the same as the outcrossing rate of the CRC (generally less than 1%), which means the gene transfer has not added a new additional risk. (2) By using historical meteorological data as an input, a regional applicable model of rice pollen dispersal and gene flow has been established, which is successively used to predict the maximum threshold distances (MTDs) of gene flow in 17 provinces of southern rice growing area in China. The feature of spatial distribution of MTDs shows: from east to west, MTDs gradually decrease; from north to south, MTDs first decrease in the hilly region and then increase again along the southeast costal region. Reason for it is that the spatial distribution is dramatically influenced by the southeast monsoon (seasonal wind direction from the southeast) and the landform structure. (3) We have artificially constructed two mixed populations of O. rufipogon with F1 hybrids of CRC/O. rufipogon derived from transgene (either Bt or bar) flow to investigate the long-term fate of the transgene integrated into common wild rice. It was found that the F1 hybrids of CRC/O. rufipogon totally disappeared within 3-5 years and the Bt or bar gene was not detectable in the mixed population. It is reasonable to speculate that the common wild rice possesses a mechanism of self-protection. (4) The effectiveness in reducing transgene flow by using flowering isolation or a protective cloth-screen in small-scale field trials of transgenic rice was studied and the results discussed. To investigate the degree of flowering synchronization of CRC and O. rufipogon populations in adjacent plantation, a survey has been conducted in Hainan, Guangdong and Guangxi provinces which allow us to establish a corresponding database. In order to further eliminate the rice transgene flow, a biological containment measure - gene split approach has been established. Based on the data obtained in this study and a survey on the internationally published data of gene flow from major crops, we have proposed to use principles of classification management and threshold-value management in the risk assessment and regulation of transgenic rice. Meanwhile, the progress and prospective of rice gene flow are also discussed in this paper.

Genetic Diversity of the Main Chinese Three-Line Hybrid Rice Parents Based on Functional Genetic Markers Related to Yield

ZHANG Tao-1, 3 , YANG Jiao-2, JIANG Kai-Feng-1, 3 , CAO Ying-Jiang-1, YANG Li-1, 3 , YANG Qian-Hua-1, WAN Xian-Qi-1, YOU Shu-Mei-1, LUO Jing-1, GAO Lei-1, LI Zhao-Xiang-1, ZHENG Jia-Kui-1, 2 , 3

Scientia Agricultura Sinica. 2014, 47(1): 11-23. doi:10.3864/j.issn.0578-1752.2014.01.002

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【Objective】 The objective of this study is to analyze the genetic diversity of the three-line hybrid rice parents based on functional genetic markers. 【Method】 Genetic diversity of 76 three-line hybrid rice parents was analyzed by 44 functional gene markers involved in QTL loci, or fine mapping, or been cloned which linked closely to rice yield trait, and these genes have been considerably reported by literature. At the same time, the genetic diversity of the above materials mentioned was also studied by 29 SSR markers with higher polymorphism, clear band pattern, reproducible, and covered in 12 chromosomes of three-line hybrid rice parents. According to claim of POPGEN32 analysis software, the PCR gel electrophoresis product data matrix was transformed into the genotype data, and the alleles (Na), effective number of alleles (Ne), percentage of polymorphic loci (P), and Nei’s genetic diversity index (He) were calculated. This study further evaluated the genetic diversity of all parents according to the deduced information of POPGEN32 analysis software. Besides, this study also calculated the genetic differentiation coefficient (Fst), Nei’s genetic distance (D), and further checked out the genetic structure and genetic relationship. NTSYS-pc2.10e software was used to calculate the genetic similarity coefficient (GS), and cluster analysis was made according to GS group and using the non-weighted average method (UPGMA), and the genetic dendrogram was mapped. 【Result】Of which 37 functional gene markers showed polymorphism and 86 total alleles loci were detected; the percentage of polymorphic loci (p) was 84.09%. While the number of effective alleles(ne) was 62.95, which accounted for 73.2%, Nei’s genetic diversity index(he) ranged from 0.049 to 0.831, and 0.650 in avarage. The genetic similarity (GS) of 76 varieties ranged from 0.323 to 0.973, and 0.650 in average. UPGMA cluster analysis showed that 76 accessions could be classified into two distinct classes of maintainer lines and restoring lines, at similarity coefficient of 0.618. The coefficient of genetic differentiation(gst) was 0.151, belonging to high variation level, and the Nei’s genetic distance (GD) was 0.185. The genetic distance within the groups was relatively small, relatively large among the taxa. A total of 72 alleles loci were detected by 29 SSR markers. UPGMA cluster analysis showed that the 76 accessions cannot be classified into two distinct classes of maintainer lines and restoring lines. Part maintainer lines clustered in a group of restoring lines, some restoring lines clustered in the maintainer line group. 【Conclusion】 The studies suggested that the functional gene markers had a high DNA polymorphisms detection efficiency, and can be used as a useful tool for their accuracy and reliability of measuring genetic diversity. The backbone parents in research showed nearer genetic relationship, higher homology sort of genetic basis. However, there still showed higher genetic differentiation between maintainer line and restoring line, suggesting that there was a higher space of using heterosis breeding in the yield of rice parents.

QTL Mapping of Oleic Acid, Linolenic Acid and Erucic Acid Content in Brassica napus by Using the High Density SNP Genetic Map

LIU Lie-Zhao, LI Jia-Na

Scientia Agricultura Sinica. 2014, 47(1): 24-32. doi:10.3864/j.issn.0578-1752.2014.01.003

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【Objective】Rapeseed oil contains many fatty acid components and the major goal in breeding and genetic research is to increase of the oleic acid content and decreases of the linolenic acid and erucic acid content. QTL mapping of oil quality related oleic acid, linolenic acid and erucic acid content in Brassica napus was made by using the high density SNP genetic map which was constructed from the high generation RIL population with the newly developed rapeseed 60K chip array. 【Method】 The high generation RIL population was derived from the cross of high erucic acid female parent GH06 and low erucic acid male parent P174. Seeds harvested from Giessen and Hohenlieth in Germany in 2008 and again in Giessen in 2009 were measured by NIRS with three technical repeats. The RIL population genotype was analyzed with the newly developed rapeseed 60K chip array. The DNA preparation and the chip preparation were processed strictly according to Infinium HD Assay Ultra manual of Illumina Inc. The SNP linkage map was constructed by using MSTmap program with minimum LOD 5.0. The reference SNP genetic map contains 2756 SNP markers, covering 1 832.4 cM of B. napus genome. QTL mapping of oleic acid, linolenic acid and erucic acid content was conducted by composite interval mapping using software Windows QTL Cartographer. 【Result】 Trait analysis indicated that the oleic acid and erucic acid contents were controlled by major genes, and their correlation coefficient was higher than -0.95 in the three different environments at significant level. Linolenic content showed a negative and positive correlation with erucic acid and oleic acid content, respectively. Two major QTL located on A08 and C03 were detected both for oleic acid and erucic acid content at the same position. In the three different environments, the major oleic acid QTL accounted for the phenotype variation from 19% to 31%, and the major erucic acid QTL effects varied from 19% to 34%, and the two major QTL for oleic acid and erucic acid content gave opposite direction of additive effect. The additive effect of the major erucic acid QTL on A08 and C03 come from the low oleic acid and high erucic acid parent GH06 with value varied from 7.6 to 9.6 in three different environments. Different QTL for linolenic acid was found across three different environments with QTL effects varied from 3% to 12%, which was coincided with that the linolenic acid was a typical quantitative trait, and was easily affected by the environment factors. The genetic map and physical map comparison discovered that the FAD2 gene and FAE1 gene located on the QTL qA05C18:3 and qA08C22:1confidence interval, respectively. 【Conclusion】 The oleic acid, linoleic acid and erucic acid content QTL was mapped with a new set of rapeseed 60K chip array, and the major QTL on A08 and C03 for erucic acid content is also responsible for oleic acid content. The mapping results are helpful to the fatty acid composition QTL comparison in different populations based on the same rapeseed 60K chip array.

Study on Characteristics of Nitrogen Metabolism in Diallel Cross Generation of Different Maize Genotypes After Silking

LIU Chun-Xiao-1, ZHAO Hai-Jun-1, DONG Shu-Ting-2, WANG Qing-Cheng-1, LI Zong-Xin-1, LIU Kai-Chang-1

Scientia Agricultura Sinica. 2014, 47(1): 33-42. doi:10.3864/j.issn.0578-1752.2014.01.004

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【Objective】 This study aimed to explore the characteristic of nitrogen metabolic in diallel cross generation of different maize genotypes after silking.【Method】Selection high nitrogen use efficiency hybrid maize plays an important role in high yield and high efficiency production of maize A full diallel cross was made with three stay-green inbred lines (Q319, CZ01, VA91) and three non-stay-green inbred lines (BM, B73, MO17) in field. The physiological traits of nitrogen metabolic in diallel cross generation were analyzed.【Result】Content of nitrogen in leaf graphed as inverted “V”-shapes after silking stage in different maize genotypes. The peak of nitrogen content in leaf occurred in filling stage. Content of nitrogen in stem-sheaf and sheath reduced with the extension of the growth duration. Content of nitrogen in leaf, stem-sheaf and sheath of stay-green (SG) inbred lines were much higher than those in non-green-stay (NSG) ones. Nitrogen accumulation before silking stage (NABS), nitrogen accumulation after silking stage (NAAS) and total nitrogen accumulation (TNA) in SG self cross were 20.00%, 82.30% and 45.23%, respectively, higher than those in NSG ones. However nitrogen translocation, nitrogen translocation rate and nitrogen harvest index in SG were 51.28%, 66.59% and 14.32% , which was significantly lower than those in NSG ones. TNA of SG was about 50% and TNA of NSG was only 40% after silking stage. Crude protein content in leaf and stem-sheaf of SG were 108.23% and 33.63%, higher than those in NSG ones. The straws were still higher valuable after harvesting. Some key enzymes to nitrogen metabolic activity such as nitrate reductase (NR) activity, glutamate synthetase (GOGAT) and glutamine synthetase (GS) in leaf of stay-green type were significantly higher than those in non-green-stay types. NR activity in leaf increased firstly and the peak occurred in filling stage after silking stage in different maize genotypes. GS activity, GOGAT activity and soluble protein content in leaf increased firstly, and the peak occurred 15 days after silking. Correlation analysis between stay-green degree, content of nitrogen, soluble protein content, GS activity, GOGAT activity and NR activity indicated that there existed significantly or highly significant positive correlations. Activities of those key enzymes on nitrogen metabolism were significantly important for nitrogen assimilation. 【Conclusion】 High nitrogen metabolism level is one of the nutrition physiological basis which makes leaves keep green for long time. Compared with the non-stay-green types, stay-green genotypes behave higher activities of key enzymes to nitrogen metabolic and higher nitrogen asorption and assimilation at late growth stage. In conclusion, character of stay-green could be used as an important agronomic trait in selecting high nitrogen efficient genotypes and inbred line.

Effect of Potassium Application Time on Starch Accumulation and Related Enzyme Activities of Sweet Potato Variety Jixu 23

LIU Hong-Juan-1, YAO Hai-Lan-1, SHI Chun-Yu-1, ZHANG Li-Ming-2

Scientia Agricultura Sinica. 2014, 47(1): 43-52. doi:10.3864/j.issn.0578-1752.2014.01.005

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【Objective】A field experiment was conducted to clarify the regulatory effects of potassium application time on starch accumulation and its physiological mechanism, starch granule distribution of storage roots of sweetpotato variety Jixu 23. 【Method】Jixu 23 (cultivar for starch use) was planted at Tai’an experimental site in Shandong Province, with randomized block design in 2008-2009 growing season. There were four treatments in all, including the control (non-potassium fertilizer, T1), potassium applied as basal fertilizer (T2), as top cover fertilizer (T3) and as top-growth peak fertilizer (T4). Samples were obtained by using periodic sampling during storage roots development. The content of starch, amylose and amylopectin of storage root were determined at different growth stages. Meanwhile the medium diameter of starch grain was determined and the volume fraction of different diameters starch granules (＞19.76 μm, 3.359-19.76 μm and ＜3.359 μm) was calculated. The activities of enzymes (SS, ADPGPPase, UDPGPPase, GBSS and SSS) related to starch synthesis were also studied. The effects of potassium application time on starch and its components, starch granule distribution and activities of enzymes related to starch synthesis in storage roots at different growth stages were analyzed.【Result】Compared with the control (T1), dry matter accumulation and starch yield of storage roots in treatments with potassium applied increased, and the increase of starch yield of T2, T3 and T4 treatments was 24.90%, 16.57%, and 9.24%, respectively. And the increase decreased when the application time delayed. The amylose content of storage root in treatments of topdressing (T3, T4) increased while amylopectin content was decreased significantly. The volume fraction of large starch granules in treatment with potassium applied as basal fertilizer (T2) was larger, and the volume fraction of medium and small starch granules in treatment with potassium applied as top-growth peak fertilizer (T4) was larger than that in the other treatments. Potassium application increased the activities of SS, ADPGPPase and UDPGPPase, and enhanced the supply capability of precursors for the synthesis of starch. And the longest action time of potassium on SS and ADPGPPase activities was 130 days, and potassium applied as basal fertilizer (T2) worked effectively mainly at early and middle stages of storage roots development, while treatments of topdressing (T3, T4) worked effectively mainly at middle and late stages of storage roots development. For UDPGPPase activity, the earlier the potassium applied, the longer action time was got. Potassium applied as topdressing (T3, T4) increased SSS activity but reduced GBSS activity of storage roots during the later expanding period. There was a significant positive correlation between total starch content and SS and ADPGPPase activities, and the significant positive correlation was also found between amylose content and GBSS and SS activities. And amylopectin content had a significant positive correlation with ADPGPPase and SSS activities.【Conclusion】Potassium applied as basal fertilizer (T2) and top cover fertilizer (T3) increased dry matter accumulation and starch yield of storage root significantly. Potassium applied as topdressing increased amylopectin content but decreased amylase content significantly. Treatments with potassium applied as basal fertilizer got a higher volume fraction of large starch granules, while treatments with potassium applied as top-growth peak fertilizer got a higher volume fraction of medium and small starch granules. The significant effects of potassium application time on amylase content, amylopectin content and starch yield were closely related with the changes of SS, ADPGPPase, UDPGPPase, GBSS and SSS activities.

Identification of Pathogen Causing Crown and Root Rot on Strawberry

HAN Yong-Chao, XIANG Fa-Yun, ZENG Xiang-Guo, ZHANG Peng, GU Yu-Cheng

Scientia Agricultura Sinica. 2014, 47(1): 53-60. doi:10.3864/j.issn.0578-1752.2014.01.006

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【Objective】The objective of this study is to identify the pathogen causing crown rot on strawberry in Wuhan, and to provide a basis for disease prevention and resistance breeding.【Method】Diseased samples from Wuhan, Hubei Province in central China were collected, pieces of infected tissue from the crowns were surface disinfested and plated on acidified potato dextrose agar (PDA). The growing edges of any fungal hyphae developing from the tissues were then transferred aseptically to another PDA plate. Isolates were characterized by colony morphology, conidial characteristics and mycelia growth rate under 18℃ in dark. One-way analyses of variance (ANOVA) were conducted to determine the significance of differences in growth rates of different isolates. According to the morphological characteristics, isolates Zhd-3, Zhd-4-1 and Zhd-5 were selected as representatives for next analysis. Pathogenicity test was carried out by inoculating conidia of the three isolates to crown of strawberry with toothpick, respectively. Conidia produced on petioles were observed by optical microscope and scanning electron microscopy. The length and width of 20 conidia of each isolate were measured. Partial actin (ACT), beta-tubulin (TUB2) and calmodulin (CAL) genes of Zhd-3, Zhd-4-1 and Zhd-5 were amplified and sequenced. The ACT, TUB2 and CAL sequences of Zhd-3, Zhd-4-1, Zhd-5, along with the 23 different representative isolates in C. gloeosporioides species complex were used for multigene loci analyses with Mega 4.1 and Neighbour-Joining method. 【Result】Fifteen isolates were morphologically identified as Colletotrichum spp.. Colony morphology of these isolates on the above of PDA plate appeared similar but significantly different on the back. The 15 isolates were divided into three groups based on the colony morphology, and isolates Zhd-3, Zhd-4-1 and Zhd-5 belonged to each group, respectively, were selected for follow-up tests and analysis. The pathogenicity tests showed that the symptoms identical to those observed in field were produced on all inoculated plants. Control plants remained symptomless. The same pathogen was reisolated from the diseased tissues. Biological tests showed that mycelial growth rates of Zhd-3, Zhd-4-1 and Zhd-5 were 0.82, 0.68 and 0.88 cm?d-1, respectively. The average size of 20 conidia was 11 μm × 3.8 μm. The biological characteristics indicated these isolates were C. gloeosporioides species complex. The result of multigene loci analysis based on ACT, TUB2 and CAL indicated that isolates Zhd-3, Zhd-4-1 and Zhd-5 were divided into one group with C. siamense strains ICMP12567, ICMP17795, ICMP18121, CBS113199 and CBS112983.【Conclusion】The results of biological characteristics and multigene loci analysis based on the sequences of TUB2, ACT and CAL are concluded that the crown rot disease of strawberry occurred in Wuhan is caused by C. siamense.

Life Tables for Experimental Populations of Frankliniella occidentalis (Thysanoptera: Thripidae) Under Constant and Fluctuating Temperature

WANG Hai-Hong-1, XUE Yao-2, LEI Zhong-Ren-1

Scientia Agricultura Sinica. 2014, 47(1): 61-68. doi:10.3864/j.issn.0578-1752.2014.01.007

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【Objective】The effects of constant temperatures on the life table parameters of western flower thrips (Frankliniella occidentalis) were well established, and the results were used as a means of projecting the growth of populations under natural condition. F. occidentalis in the wild, however, are not exposed to constant temperatures as they are faced with temperature variation on a daily basis. In this study, life tables of F. occidentalis under constant temperature (CT) and daily fluctuating temperatures (DFT) were compared, and the accuracy of population occurrence dynamics in natural condition based on data from CT were explored. 【Method】Developmental, survival, and fecundity rate data were analyzed using the age-stage, two-sex life table of F. occidentalis reared on bean pod were constructed under constant temperature (CT, 26℃) or daily fluctuating temperatures with same mean (DFT, 20-32℃). As the hatch rate of eggs varies with maternal age, the age-specific fecundity was calculated based on the numbers of hatched eggs to reveal the biological characteristics of F. occidentalis accurately. Means and standard errors of population growth parameters were calculated using the bootstrap method. The Mann-Whitney test (U test) was used to evaluate the differences in the population parameters, development times, and fecundities of F. occidentalis reared under CT and DFT.【Result】There were significant differences in pre-adult developmental time, total preoviposition period, pre-adult survival and life table parameters between F. occidentalis reared under CT and DFT (P＜0.05). However, no significant difference was found in adult longevity and fecundity between F. occidentalis reared under CT and DFT. The pre-adult developmental duration of F. occidentalis reared under CT (11.86 d) was significantly longer than those under DFT (11.36 d). The total preoviposition period of F. occidentalis reared under CT (12.50 d) was significantly longer than those under DFT (11.37 d). The pre-adult survival of F. occidentalis reared under CT (0.48) was significantly higher than those under DFT (0.44). The numbers of eggs laid per female were 35.38 and 34.74 for F. occidentalis reared under CT and DFT, respectively. The intrinsic rate of increase (r), finite rate (λ) and net reproductive rate (R0) of F. occidentalis reared under CT were 0.121 d-1, 1.129 d-1 and 7.538, respectively, while those reared under DFT were 0.127 d-1, 1.135 d-1 and 8.831, respectively. The population growth of F. occidentalis reared under DFT was quicker than those reared under CT. 【Conclusion】The results indicated that by using CT, one could under- or over-estimate the values of numerous life-history traits compared to more natural DFT conditions. This inaccuracy may in turn reduce the accuracy of population surveillance and downstream applications for the timing of integrated pest management.

Influence of Spray Method on the Deposit and Distribution of Spray Droplets in Rice Field

XU De-Jin, GU Zhong-Yan, XU Guang-Chun, XU Xiao-Long

Scientia Agricultura Sinica. 2014, 47(1): 69-79. doi:10.3864/j.issn.0578-1752.2014.01.008

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【Objective】The objective of this study is to analyze the influence of spray method on the deposit and distribution of pesticide within the rice population, and to put forward reasonable spay technology and improve the utilization ratio of pesticide in rice field. 【Method】The solution of ponceau-G was sprayed by manual sprayer with a hollow cone nozzle upon rice plant and mist sprayer with pneumatic nozzle down 45° tilt angle at rice tillering, booting and flowering stages. The ponceau-G deposited on the microslides, which respectively showed the obverse and reverse of rice leaves and the vertical stem of plant, were collected and measured.【Result】There was a significant difference among the deposition amounts of penceau-G sprayed by different methods at different sites within the field rice population at different growing stages. The deposit rate of ponceau-G was manifested with a distribution trend of upper layer＞middle layer＞lower layer. At the rice tillering stage, manual sprayer was applied and the deposition of ponceau-G on 13.3% of microslides was close to the average value, the deposition of ponceau-G 52.9% of microslides was lower than the average value, and 33.75% was higher than the average value. At the rice tillering stage, mist sprayer with pneumatic nozzle down 45° tilt angle was applied and the deposit rate of ponceau-G on 20.4% of microslides was close to the average value, 45.4% was lower than the average value, and 34.2% was higher than the average value. At the booting and flowering stages, manual sprayer was applied and 33.3% and 28.1% of microslides did not have any deposition of ponceau-G. When mist sprayer with pneumatic nozzle down 45° tilt angle was applied, there was 13.9% and 5.0% of microslides did not have any ponceau-G deposition. The deposition of only 6.4% and 11.7% of microslides was close to the average value at booting stage for manual sprayer and mist sprayer. At the flowering stage, the proportion was 7.2% and 17.2%, respectively. For manual sprayer, ponceau-G mainly deposited on the microslides expressing obverse of leaves and the amounts were, respectively, 66.3%, 85.1% and 84. 9% of total deposits on microslides in three rice stages, among which the amount of ponceau-G on the microslides from upper layer of plant were, respectively, 38.7%, 42.2% and 45.6% of total deposition. There were litter deposition on the microslides expressing the reverse of rice leaves and the vertical stem of plant, especially on some microslides put in the base of rice plant, the amount of ponceau-G was zero at rice booting and flowering stages. For mist sprayer, the deposition on the microslides showed the reverse of rice leaves and the vertical stem of plant was more than that of manual sprayer. However, the ponceau-G on the microslides showing obverse of leaves were still 50.5%, 50.6% and 53.1% of total deposits, among which the ponceau-G on the microslides from upper layer of plant was, respectively, 32.3%, 27.9% and 31.5% of total deposition.【Conclusion】If the average of total deposition on each microslides was the pesticide dose controlling pest effectively, there were shortage and waste of dose within rice population at the same time. The fog drip of manual sprayer spraying mainly deposited on the obverse of rice leaves of plants’ upper leaves. The deposition rate accounts for above 1/3 of the total deposition amount, while the deposition rate was few on the reverse of leaves and the vertical stem, especially on the plants’ base leaves and vertical stem, on which the deposition rate was nearly zero. When mist sprayer with pneumatic nozzle down 45° tilt angle was applied, the deposition rate of fog drip on the obverse of rice leaves of plants’ upper leaves was more than 1/4 of the total deposition amount. However, when the application volume was less than that of manual sprayer conducting coping spraying, each layer of the reverse of leaves and the vertical stem was demonstrated a significantly higher deposition rate than manual sprayer. It illustrates that spraying with the mist sprayer with pneumatic nozzle down 45° tilt angle, pesticide droplets could be delivered to middle and lower layer and deposited on rice stem and reverse side of leaves.

Cloning and Resistance Verification of a Target Gene of Glyphosate from Klebsiella pneumoniae S001

ZHANG Chun-1, WU Dan-Dan-1, 2 , FENG Li-1, TIAN Xing-Shan-1, GUO Ai-Ling-2

Scientia Agricultura Sinica. 2014, 47(1): 80-89. doi:10.3864/j.issn.0578-1752.2014.01.009

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【Objective】Due to the widespread use of glyphosate in agricultural production, glyphosate has become the preferred object on transgenic crops resistant to herbicides. Finding glyphosate resistance genes is a primary issue in the study of transgenic crops resistant to glyphosate. There are various kinds of microorganisms in nature with rich genetic resources, so this study intends to screen and identify high glyphosate-resistance bacterium strains from field soil samples of Guangdong area, then clone the target gene of glyphosate from the strain and test its glyphosate-resistance level, in order to obtain high glyphosate resistance gene resources for research of glyphosate resistant transgenic crops.【Method】The gradient dilution glyphosate selected culture method in the isolation of high glyphosate-resistance bacterium from field soil samples of Guangdong area was applied. Identification of the strain species according to the microscopic observation, gram staining and 16 S rDNA sequence analysis results. Using RT-PCR method to clone the strain’s target gene of glyphosate, and analyse the basic characteristics of aroAS001 sequences by sequence alignment and phylogenetic tree. The aroAS001 variant performed site directed mutagenesis by overlapping PCR method to obtain aroAS001-mut gene fragments. The aroAS001 and aroAS001-mut fragments were transferred into defective Escherichia coli DH5α/△aroA strains, respectively, to detect the resistant levels of glyphosate. 【Result】 A high glyphosate-resistance bacterium strain was isolated, and it was identified as Klebsiella pneumoniae by morphological and molecular biology methods, named kpS001 strain. The target gene of glyphosate from kpS001 strain named aroAS001 was cloned. Sequence analysis showed that the amino acid sequence of the gene encoded had a typical Class I EPSPS features, and there was a single amino acid different from with another K. pneumoniae strain’s aroA. After obtained the aroAS001-mut fragments by overlap PCR, aroAS001 and aroAS001-mut fragments were transferred into the defective E. coli DH5α/△aroA strains, respectively, to detect the resistant levels of glyphosate. Compared with the control strains, the recombinant strains containing aroAS001 and aroAS001-mut were able to grow normally in the culture medium containing less than 200 mmol?L-1 glyphosate, however, as glyphosate concentration increasing, the growth state of the recombinat strains gradually suppressed, while the concentration of glyphosate increased to 350 mmol?L-1, the growth was completely suppressed. 【Conclusion】The K. pneumoniae S001 is a high glyphosate-resistance bacterium, the target gene of glyphosate aroAS001 belongs to the Class I EPSPS and shows a significant glyphosate-resistance characteristics, it could be used as potential gene materials in transgenic glyphosate-resistant crop studies.

Study on Potassium Use Efficiency and Apparent Soil Potassium Balance Under Long-Term Rice-Barley Rotation

TANG Xu, JI Xiao-Jiang, LI Chao-Ying, WU Chun-Yan, YANG Sheng-Mao, LIU Yu-Xue, 吕Hao-Hao , CHEN Yi

Scientia Agricultura Sinica. 2014, 47(1): 90-99. doi:10.3864/j.issn.0578-1752.2014.01.010

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【Objective】The potassium (K) natural productivity from soil, K fertilizer use efficiency and apparent soil K balance were investigated to provide a theoretical basis for the sustainable management of K element in farmland. 【Method】The three fertilizer treatments used were control, NP and NPK, representing various combinations of N, P and K fertilizer applications. Long-term (over 20 years) experiments with barley-rice crop rotation were conducted to investigate the response of crop aboveground biomass, yield, the K content in grain and straw to different application treatments. 【Result】The results indicated that the grain yields on average were 8.24 and 9.22 t?hm-2 per year in barley-single crop rice and barley-early rice-late rice rotation cropping system without application fertilization for 20 years, which accounted for 75.6% and 71.9% of that in NPK treatments in the corresponding rotation system. The contribution of application fertilizers increased crop grain yields by 24.4% to 28.1%, and the contribution of K fertilizers increased crop grain yields by 11.8% to 14.0% in barley-single crop rice and barley-early rice-late rice rotation cropping system, respectively. The contribution of K fertilizers increased grain yields for barley (17.9%) was 36.1% higher than that for rice. The annual K uptakes from CK treatments accounted for 59.1% and 58.3% of that in NPK treatments. The K in straws accounted for 81.7% and 74.2% to 87.6% of total K uptake for barley and rice, respectively. The indigenous K supplying capacity in barley season was lower than that in rice season. The annual indigenous K supplying capacity for barley on average was 69.8%, which were 15.5%, 19.7% and 19.4% lower than that for early rice, late rice and single crop rice, respectively. The indigenous K supplying capacity in barley season decreased significantly over cultivation time at an average rate of 1.6% per year. But there was no significant change of the indigenous K supplying capacity in time during rice cropping. The indigenous K supplying capacity in rice season still was more than 80% after 20 years cropping. The internal K use efficiency was decreased because of K application. But there was no significant change of internal K use efficiency among treatments in time. Under balanced fertilization with fixed N, P and K inputs, the internal use efficiencies for barley, early rice, late rice and single-crop rice were 42.4, 44.7, 41.3 and 44.8 kg•kg-1, respectively. The partial factor productivity in NPK treatment was 83.5, 79.7, 71.6 and 69.7 kg•kg-1, respectively. The K use efficiency in barley season was higher than that in rice season. The apparent K recovery efficiency was 29.5% and 41.5% in barley-single crop rice and barley-early rice-late rice rotation system, respectively. Crop continuous uptake caused a net K lost of 148.2 and 182.7 kg•hm-2 per year in the soils without application of fertilizer in barley-single crop rice and barley-early rice-late rice rotation cropping system, respectively. Moreover, the application of nitrogen and phosphate fertilizer accelerated soil K depletion, the extra K amount of 57.8 and 65.0 kg•hm-2 was taken up by crop. Although K deficiency in soil could be relieved by K application, a net K lost of 146.5 and 85.5 kg•hm-2 per year in soils received K fertilizer from barley-single crop rice and barley-early rice-late rice rotation cropping system. 【Conclusion】There was a very strong capacity to provide K element from soils during rice growing season. But the K fertilizer use efficiency in barley season was higher than that in rice season. There was a still net K loss from soil with K application rate of 187.5 kg K2O•hm 2 per year. So a great attention should be paid to the apparent soil K balance.

Characterization of Soil Salinization Based on Canonical Correspondence Analysis Method in Gansu Yellow River Irrigation District of Northwest China

YANG Si-Cun-1, PANG Huan-Cheng-2, WANG Cheng-Bao-1, LI Yu-Yi-2, HUO Lin-1, JIANG Wan-Li-1

Scientia Agricultura Sinica. 2014, 47(1): 100-110. doi:10.3864/j.issn.0578-1752.2014.01.011

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【Objective】The objective of this study was to investigate the spatial distribution characteristics and quantitative relations of total salt content (TS), pH value and ion components so as to evaluate the relationship and correlation between them and their influences on types and distribution of saline-alkali land in Gansu Yellow River Irrigation District, Northwest China. 【Method】Take the typical irrigation area of Xingdian and Jingdian as research soil sampling area with sampling sites fully covered the newly reclaimed saline-alkali wasteland in the upper reaches, the mildly salinization of cultivated land in the middle reaches, the moderately severe salinization of cultivated land in the lower reaches and the salinization abandoned cropland in the whole irrigation area. And given more consideration to the differences between the types (sulfate type, chloride-sulfate type, sulfate-chloride type) and the degrees (mild, moderate, severe) of soil salinization when sampling. The conventional statistics method was used to evaluate the soil profile distribution of TS, pH and salt ion. The canonical correspondence analysis method was used to evaluate the quantitative relationships of TS, pH and the contents of salt ion, and the correlation coefficients, eigenvalues, cumulative percentage variance of TS, pH, salt ions between axis 1 and axis 2 of CCA in various soil layers. 【Result】 The irrigated soils of Gansu Yellow River Irrigation District are mild to severe salted, the salt content of 0-40 cm soil layer decreased with soil depth increase while the salt content of 40-80 cm soil layer increased with soil depth increases. The cation and anion in soil profile are Na+ and SO42 respectively -with pH value vary between 7.93 and 9.98. K+ and HCO3-, Ca2+ and Mg2+ are well linked in all soil layers, the correlation between Na+ and Cl- are enhanced with increment of soil depth, SO42- and Na+ are correlated negatively, the correlation between K+ and Na+ as well as SO42- , Cl- and HCO3 are not stronger in all soil layer-. At 0-100 cm soil profile, the spatial distribution of pH is mainly controlled by HCO3-, TS is mainly controlled by Na+, Cl- and SO42-, respectively. Different soil layers have diverse characteristics of soil salinization from that of the whole soil profile, the spatial distribution of pH and TS is mainly controlled by SO42- in 0-20 cm soil layer, by Cl- and Na+ in 20-40 cm soil layer, by Mg2+ in 60-80 cm soil layer, by Mg2+ and Cl- in 80-100 cm soil layer, by Ca2+, Mg2+, SO42- and HCO3- together in 40-60 cm soil layer individually. Axis1 and axis 2 of CCA reflects the changes of TS and pH value, respectively. The eigenvalues and cumulative percentage vary from 0.53 to 0.70 and 38.7% to 57.7% of axis 1, respectively, while that vary from 0.36 to 0.64 and 30.6% to 44.7% of axis 2 correspondingly. 【Conclusion】There are two processes of surface salt accumulation and bottom salt accumulation profiles in the saline cultivated land of Gansu Yellow River irrigation District. Newly reclaimed saline-alkali wasteland in the upper reaches and mild salinization of cultivated land in the middle reaches are given priority to surface accumulation profiles, and mild salinization of cultivated land in the middle reaches and moderately severe salinization of cultivated land in the lower reaches are given priority to bottom accumulation profiles. The salt content of 60-80 cm argillaceous cemented sand layer is as high as 93.7 g•kg-1, which has an important influence on the migration of water and salt and the distribution of salt ions, and is one of the main causes to lead soil to secondary salinization in the irrigation area. TS, Na+ and SO42- are the main factors that determine the type and distribution of saline-alkali land in Gansu Yellow River Irrigation District. Meanwhile, the canonical correspondence analysis method can intuitively give the relationship among various salt ions and between TS, pH and salt ion in two-dimensional ordination diagram of CCA by the length of the arrow, the quadrant, the angle between arrows, the distance between arrows and the triangle symbol, etc. And it also can determine the main controlling factors that influence the characters of different layers and the main factors that affect the type and distribution of saline-alkali land by using correlation coefficients, eigenvalues, cumulative percentage between TS, pH, salt ions and axis1/axis2 of CCA in various soil layers, so that, it is considered to be the more efficient scientific method for evaluation of the characteristics and improvement of saline-alkali land in Yellow River Irrigation Area.

EST-SSR Fingerprinting of Fifty Cabbage Representative Varieties from China

WANG Qing-Biao, ZHANG Yang-Yong, ZHUANG Mu, YANG Li-Mei, LIU Yu-Mei, 吕Hong-Hao , FANG Zhi-Yuan

Scientia Agricultura Sinica. 2014, 47(1): 111-121. doi:10.3864/j.issn.0578-1752.2014.01.012

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【Objective】In this study, the method of cabbage DNA fingerprint was drawn, and fifty cabbage representative varieties from China were fingerprinted with EST-SSR primers to provide reference for variety distinctness, authenticity, and purity identification. 【Method】First, EST-SSR primers were screened by using the technologies of 6% denaturing polyacrylamide gel electrophoresis and six cabbage varieties that come from different ecogeography. The length of amplified fragments was detected on DNA Analyzer platform using four fluorescent-labels (TAMRA, HEX, ROX and 6-FAM) in 5′end of forward primer, and then defined the reference variety for every alleles. Total twenty core primers were used to establish fifty cabbage varieties SSR fingerprinting, and for ‘Zhonggan 21’ variety identification. 【Result】 Six cabbage varieties of different resources were used to screen 978 EST-SSR primers, out of 128 polymorphic primers were obtained according to the PCR bands stability, high polymorphism information content (PIC), easy discrimination of different alleles and even distribution of molecular markers on each chromosome, and 20 pairs of primers were selected to detect a total of 58 alleles at 20 loci, with 2.22 loci per chromosome and 2.9 alleles per locus on average. The PIC values varied among the primers ranging from 0.34 to 0.76. The length of amplified fragment varied in the range of 143-296 bp. The maximum number of alleles for each primer pairs of BoE607 and BoE723 was five. Fingerprinting database of 50 cabbage representative varieties from China was established with 20 pairs of core primers. The authenticity of ‘Zhonggan 21’ was identified by artificial simulated population and these results were identical with that made from field investigation.【Conclusion】Twenty pairs of core primers were selected and used to establish DNA fingerprint database of 50 cabbage representative varieties from China, and the authenticity of ‘Zhonggan 21’ was identified by artificial simulated population and the accuracy was 100%.

Molecular Mapping and Candidate Gene Analysis of Black Fruit Spine Gene in Cucumber (Cucumis sativus L.)

LIU Shu-Lin-1, GU Xing-Fang-1, MIAO Han-1, WANG Ye-1, YiqunWeng2 , ToddCWehner3 , ZHANG Sheng-Ping-1

Scientia Agricultura Sinica. 2014, 47(1): 122-132. doi:10.3864/j.issn.0578-1752.2014.01.013

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【Objective】Cucumber (Cucumis sativus L.) is an important fruit vegetables. Fruit quality is always getting more attention in cucumber breeding program. Fruit quality includes inner quality and exterior quality, and fruit exterior quality of cucumber has important influences on its commodification. Spine color is one of the important fruit quality traits in cucumber. The clarification of the inheritance and identification of molecular markers for the fruit spine color gene will provide a theoretical basis for breeding of fruit quality and lay a foundation for fine mapping and gene cloning. 【Method】 Cucumber inbred lines GY14 with white fruit spines and NC76 with black fruit spines were used as the experiment materials for genetic analysis and gene mapping for black fruit spine in this study. Bulked segregation analysis (BSA) was performed in the F2 population using 2112 SSR markers. The sequence and re-sequencing information of 9930 and 100 core germplasms were used to develop new SSR and Indel markers in the primary mapping region of the black spine color gene (B). JoinMap 4.0 and MapInspect software were employed to construct a linkage map for the B gene with SSR markers. Bio-informatics was adopted to predict candidate genes in the genomic region harboring the B gene. A set of 156 recombinant inbred lines (RILs) were used to test the veracity for marker-assisted selection (MAS) of flanking molecular markers linked to the B gene. 【Result】Genetic analysis showed that the trait of black fruit spine in NC76 was qualitative, and a single dominant nuclear gene (B) controlled this trait. Black was dominant to white. In the primary genetic mapping of the B gene, eight SSR markers were screened to be linked with the black fruit spine color locus. The B gene was mapped on the chromosome 4 (Chr.4) of cucumber. The closest linked marker SSR22231 was 10.8 cM away from B. A total of 212 pairs of new SSR primer and 25 pairs of Indel primer were developed based on the sequence information in the primary mapping region of B. Fourteen SSR markers and one Indel marker were identified to be linked with the B gene after analysis of the F2 mapping population using these new developed molecular markers. The two closest flanking markers SSRB-181 and SSRB-130 were 2.0 and 1.6 cM away from B, respectively. The physical distance between SSRB-181 and SSRB-130 was 422.1Kb containing 60 predicted genes. Csa4G003095 and Csa4G001690 were the most possible candidate genes for the black fruit spine trait. The probability of the two flanking SSR markers to predict the spine color for marker-assisted selection (MAS) breeding was 96.8% and 96.2%, respectively. And the accuracy rate for SSRB-181 to predict the black spine color in MAS breeding was 100%.【Conclusion】The black fruit spine trait of NC76 is controlled by one dominant nuclear gene (B). It is located on the Chr.4 of cucumber delimited in a physical distance of 422.1 Kb. The two closest flanking markers SSRB-181 and SSRB-130 could be used in the MAS breeding program. The results in this study will be of great benefit to fine mapping and gene cloning for the B gene and lay a good foundation for cucumber MAS breeding.

The Structure and Single Nucleotide Polymorphism Analysis of Chalcone Synthase Genes in Tea Plant (Camellia sinenesis)

ZHANG Li-Qun-1, WEI Kang-1, WANG Li-Yuan-1, CHENG Hao-1, LIU Ben-Ying-2, GONG Wu-Yun-1

Scientia Agricultura Sinica. 2014, 47(1): 133-144. doi:10.3864/j.issn.0578-1752.2014.01.014

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【Objective】 The objectives of this study were to determine the structures of CHS genes in Camellia sinensis (CsCHS) by obtaining gDNA sequences of these genes, and to analyze single nucleotide polymorphism. Besides, association analysis was also carried out in order to find potential SNP sites in CsCHS which would influence the polyphenol content in tea plant.【Method】 Based on CsCHS sequences uploaded to NCBI, specific primers were designed using primer 3.0 software. Genomic DNA and cDNA of tea leaves were used as templates in polymerase chain reaction (PCR) to obtain gDNA and cDNA sequences of CsCHS1, CsCHS2 and CsCHS3, respectively. Gene structures were determined through blasting gDNA and cDNA sequences. The putative amino acid sequences were analyzed by bioinformatics softwares, such as Compute pI/Mw, SOPMA, and so on. Single nucleotide polymorphism of CsCHS were analyzed in 57 cultivars with great variation polyphenol contents. In order to obtain the coding region of CsCHS genes, PCR reactions with specific primers were carried out by using cDNA of individual tea cultivars as template. TASSEL software was introduced in association analysis.【Result】 cDNA sequences of CsCHS1, CsCHS2 and CsCHS3 were 1 277 bp, 1 320 bp and 1 242 bp, respectively. And an open reading frame (ORF) of 1 170 bp was found in each CsCHS gene. gDNA sequences of CsCHS1, CsCHS2, and CsCHS3 were 1 600 bp, 1 330 bp and 1 607 bp, respectively. By comparing gDNA and cDNA sequences of each CsCHS gene, combined with GT-AG rule, it was determined that CsCHS1 and CsCHS2 have two exons and one intron, respectively. And the intron in CsCHS1 is 323 bp, compared with 356 bp in CsCHS3. While no interruption region was found in CsCHS2, this might prove there is no intron in CsCHS2. Deduced amino acid sequences analysis suggests that identity of amino acid sequences are 92.6%-95.4%. All of the conserved amino acids found in CHS protein subfamily were also found in these deduced sequences. Furthermore, bioinformatics analysis showed highly similarities among CsCHS1, CsCHS2 and CsCHS3 protein structures. There are 71 SNP sites in CsCHS1’s coding region, SNP frequency was 1SNP/16.48 bp, and no Indel was found. A total of 55 SNP sites were found in CsCHS2’s coding region, suggesting one SNP in every 21.27 bp. And the nucleotide diversity (π) in CsCHS1 (0.01088) was significantly higher than that of CsCHS2 (0.00530). By correlation analysis, two SNP sites were positioned thought to be related to polyphenol content in tea plant in CsCHS1 and 4 in CsCHS2. No further analysis referring to CsCHS3 was carried out due to low success rate of PCR reaction.【Conclusion】 Both CsCHS1 and CsCHS3 were determined to be conserved CHS genes, and bioinformatics analysis of protein structures results show that CsCHS1, CsCHS2 and CsCHS3 are similar. CsCHS1 and CsCHS3 are active, there should be hot spots of mutation in their coding regions.

Effects of Different Pre-slaughter Fasting Time on Mutton Quality

XIA An-Qi, LI Xin, CHEN Li, HE Fan, LIU Jin-Kai, WANG Zhen-Yu, NI Na, ZHANG De-Quan

Scientia Agricultura Sinica. 2014, 47(1): 145-153. doi:10.3864/j.issn.0578-1752.2014.01.015

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【Objective】Pre-slaughter fasting is defined as duration of food and water withdrawal during loading, unloading, transport and control during pre-slaughter lairage period. Impropriety of pre-slaughter handling decreases carcass yield, affects eating quality and technical characteristics of meat. The objective of this study was to investigate the effects of different pre-slaughter fasting time on quality of mutton and determine how long fasting time is suitable for production of high quality mutton.【Method】Thirty six-month old sheep were divided into 3 groups fasting for 0 h(control group), 12 h and 24 h. The eating quality (pH value, meat colour, shear force and water holding capacity), sensory quality (odour, tenderness, juiciness and overall liking), hygiene quality (aerobic plate count and coliforms), glycogen concentration, sarcomere length and protein degradation during post-mortem were analyzed.【Result】Fasting for 24 h resulted in higher pH value than 12 h and control group at 0 h, 45 min and 4 h post-mortem. As aging time extended, pH value at 24 h and 48 h post-mortem had no significant difference (P＜0.05). The group fasting for 24 h had lower cooking loss than 12 h and control group (P＜0.05). There was no significant difference among three groups in L*, a*, b*,ΔE, drip loss, shear force and sensory characteristics of odour, tenderness, juiciness and overall liking of the mutton (P＞0.05). Aerobic plate count and coliforms of the mutton from different fasting time groups were all under the range of national standard regulations at 24 h post-mortem. Aerobic plate count of mutton had no significant difference among three treatments. Coliforms of the mutton were decreased as the fasting time increased. Fasting treatments caused the decrease of enumeration of coliforms compared to control group. There was no significant difference in glycogen content of mutton at 0 h, 45 min, 4 h, 8 h, 12 h, 24 h and 48 h post-mortem among three groups. The degree of glycolysis increased but the glycogen content decreased as the extension of aging time. With the increase of pre-slaughter fasting time, sarcomere length increased. Fasting for 24 h resulted in larger sarcomere length than 12 h and fasting for 12 h was higher than 0 h (P＜0.05). The degree of myofibrillar protein degradation in different fasting treatments was consistent at 0 h post-mortem. There was no apparent bands appeared or disappeared. All groups had 27 kD fragment appeared at 24 h post-mortem. The appearance of the 27 kD was darker in fasting for 12 h and 24 h treatments than the control. It was shown that fasting for 24 h and 12 h resulted in higher protein degradation degree than the control group.【Conclusion】Fasting for 12 and 24 h increased the hygiene quality and accelerated meat myofibrillar protein degradation. Fasting for 24 h decreased cooking loss. Fasting for 12 and 24 h had no significant effects on pH value, glycogen content and other quality characteristics of mutton. Compared with the control group, fasting for 12 and 24 h had positive effects on mutton hygiene quality but no effect on eating quality and sensory quality.

Inhibition of PI3K/AKT Pathway Suppressing Porcine Skeletal Muscle Sattelite Differentiation Through Activation of FoxO1 Transcription Factor

SHI Xin-E, WU Guo-Fang, SONG Zi-Yi, LU Hong-Chao, JIA Long, ZHU Jia-Yu, YANG Gong-She

Scientia Agricultura Sinica. 2014, 47(1): 154-160. doi:10.3864/j.issn.0578-1752.2014.01.016

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【Objective】Skeletal satellite cells are activated by some specific stresses such as development and trauma, and differentiate and form myotubes to participate in the development or repair of skeletal muscle. FoxO1 negatively controls the genesis of skeletal muscle, but the molecular mechanisms by which FoxO1 funcions in the differentiation of satellite cells have not been reported so far. This experiment was conducted to explore the effects of FoxO1 on porcine skeletal muscle satellite cells differentiation, aiming to provide new theoretical reference for further research. 【Method】Extensor digitorum longus of 1 to 3-day-old piglets were used to isolate the skeletal muscle satellite cells and the cells were observed and pictures were taken by inverted microscope on day 2, day 4 and day 6, respectively. The cells were stained by immunofluorescence staining and DAPI nuclear staining on day 8 of differentiation, and observed under a fluorescence microscope. Meanwhile, the medium was replaced with differentiation medium containing 50 nmol•L-1 wortmannin (wortmannin, WM) differentiation medium when the cells density reached 70% -80% confluence, the cells were collected on day 0, day 4, and day 8, respectively. Total RNA and total protein were extracted, and Real-time qPCR and Western blotting were performed to measure the alterations in the expression of FOXO1 and myogenic differentiation marker genes caused by WM supplement. 【Result】Porcine skeletal muscle satellite cells became adherent to the dish bottem, spindle-shaped on day 2. Cell number increased on day 4 and some cells started to fuse. On day 6, cell started to grow with directivity. On day 8, cell further fused to form myotubes, further fused to form myotubes. There was no significant difference in mRNA expression level of FoxO1 between WM treatment group and the control (P＞0.05), unphosphorylated FoxO1 increased significantly (P＜0.05) with WM treatment, whereas phosphorylation level of FoxO1 dropped drastically (P＜0.05). Although on day 8 the cells displayed an alveolate morphology after treated with WM, they failed to show directional growth and formation of myotubes. Moreover, Western blotting results demonstrated that WM decreased the protein level of MyoD (early myogenic marker), MyoG (middle-stage marker), and MyHC (late marker) significantly.【Conclusion】Results of the study suggest that inhibition of PI3K signaling pathway by WM blocks results in FoxO1 phosphorylation, suppression of porcine skeletal muscle satellite cell differentiation, delay of the formation of myotubes, and down-regulation of myogenic differentiation marker genes, such as MyoD, MyoG, and MyHC. Take together, blockade of PI3K signaling pathway suppresses porcine skeletal muscle satellite cell differentiation through the activation of FoxO1.

GDF-10 Gene Polymorphism and Its Association with Production Traits in Gannan Yak

LI Tian-Ke-1, 2 , LIANG Chun-Nian-1, 2 , LANG Xia-1, 2 , PEI Jie-1, 2 , WU Xiao-Yun-1, 2 , CHU Min-1, 2 , QIN Wen-1, 2 , YAN Ping-1, 2

Scientia Agricultura Sinica. 2014, 47(1): 161-169. doi:10.3864/j.issn.0578-1752.2014.01.017

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【Objective】Growth differentiation factor-10 (GDF-10) is a member of the TGF-beta super family and the bone morphogenetic protein family (BMP). It is originally identified by PCR using degenerate oligonucleotides based on the BMP-3 sequence. Studies suggested that the GDF-10 gene plays a key role in skeletal morphogenesis. The objective of this study was to identify the polymorphisms of GDF-10 gene and their correlation with production traits was investigated for the purpose of the breeding of Gannan yak. 【Method】 Thirty samples were randomly selected from genomic DNA of 298 Gannan yak to construct DNA pool. Nine primers were designed to amplify the exons 1, 2 and 3 of GDF-10 gene. Effective PCR production was purified by DNA gel extraction kit and direct sequencing subsequently. BLAST and Chromas were used to screen the SNPs in GDF-10 gene by the wave height of sequencing map. The genotypes were determined by high-resolution melting curre (HRM) and the frequency of alleles was estimated. The association between genotypes and body measurement was evaluated by software SPSS13.0. Three SNPs (T132C, G672T and G1204A) of GDF-10 gene were identified in the population, which were at extrons 1,extron 2, and extron 3, respectively. The PHASE and SHEsis softwares were used to analyze matching chain disequilibrium and haplotype analysis, respectively. The SPSS17.0 software was used for association analysis. 【Result】Three SNPs, 12116 (G/A), 12152 (C/T) and 13041 (T/C) were discovered in exon 3. Through population genetics analysis, the results showed that three locals were at low polymorphic status (PIC＜0.25). The ?2 tests showed that the locus of 13041 (T/C) was not all in the status of Hardy-Weinberg equilibrium (P＜0.05), the other two loci were all in the status of Hardy-Weinberg equilibrium (P＞0.05). Analysis of matching chain disequilibrium and haplotype analysis show that there is a weak chain of balance between the three locals. Analysis of association of polymorphism with body measurement trait at all mutation loci show that there were significant effects on body height, body length, weight and herrt girth (P＜0.05 or P＜0.001) and there is no effects on tube girth (P＞0.05). Seven haplotype combinations were found in 298 Gannan yak, the ATC and ATT have significant effects on body measurement traits and weight. 【Conclusion】 The results suggest that three SNPs and two haplotype combinations might have potential effects on production traits in the above mentioned yak populations and could be used for marker-assisted selection.

Study on Inhibitory Effect of the Swainsonine from Alkaloid of Astragalus strictus Grah．Ex Bend on Bovine Viral Diarrhea Virus

HAO Bao-Cheng-1, 2 , WU Fan-Lin-2, XING Xiao-Yong-2, XIANG Hai-Tao-2, WEN Feng-Qin-2, WANG Xue-Hong-1, QUAN Xiao-Di-1, 2 , HU Yong-Hao-2, LIANG Jian-Ping-1, 2Ping-1 , 2

Scientia Agricultura Sinica. 2014, 47(1): 170-181. doi:10.3864/j.issn.0578-1752.2014.01.018

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【Objective】 Locoweed is one of the poisonous weeds in western China that influences the healthy development of grassland and animal husbandry. Locoweeds mainly belong to Astragalus and Oxytropis genera. As a serious lack of forage in winter season, livestock such as cattle and sheep are forced to feed them. Locoweed can decrease the production performance, toxicity and death of animals, and the direct economic loss of the grassland ecological and animal husbandry is about billions of dollars, which is the most serious poisonous weed. At present, the economic losses of animal death caused by locoweed poisoning continues to soar. Swainsonine is the main toxic component and believed to be the cause of animal locoweed poisoning. Locoweed is widely distributed and is rich in resources, and how to improve the grassland ecology and use of locoweed is becoming the topic of research subject and direction. In recent years, with the study and expansion of locoweed, people have found that swainsonine not only has a good anti-tumor effect, also it can be used as an immunomodulator, anti HIV and anti proliferation inhibitor, anti viral and cell protective agent and other drugs. At present, the domestic research on swainsonine anti-tumor and regulation of immunity is hot, However, no research reports are found in antiviral activity and mechanism of action of swainsonine up to the present. This experiment was designed to investigate the anti-virus mechanism of the swainsonine from alkaloid of Astragalus on bovine viral diarrhea virus (BVDV), and to make clear of the antiviral effect in vitro, it will provide references for the screening of anti BVDV drugs. 【Method】 Using cell culture techniques, and combined with CPE observation method and MTT colorimetric method, different concentrations SW toxicity with bovine kidney primary cells (Madin-Darby Bovine Kidney Cells, MDBK) were detected, the concentration of the drug’s safety and TD50 was determined. They were measured by four administration routes of virus before swainsonine, virus after swainsonine, virus-and-swainsonine after 2 hours, and virus-and-swainsonine after swainsonine, which are the effect of different concentrations of SW against blocking effect on BVDV invasion, replicatting inhibition, directly killing effect and combining effects, respectively. The treatment indexes under different interaction modes were also calculated.【Result】It was found that the TCID50 of BVDV-MDV is 10-4.7. SW was not toxic to MDBK when its concentration was below 0.256 μg•mL-1, and TD50 is 2.512 μg•mL-1. Under the way of virus after SW, IC50 is 2.399 μg•mL-1, TI is 1.05. Under the way of SW after virus, IC50 is 1.018 μg•mL-1, TI is 2.47. Under the way of 2 h after mixed virus with SW, IC50 is 1.205 μg•mL-1, TI is 2.08. Under the way of synthetic action, IC50 is 0.782 μg•mL-1,TI is 3.21. After the TI of different modes of action was compared, it showed that the comprehensive effect on BVDV of SW (65.29%，P＜0.01) and the inhibition effect of SW on the reproduction of BVDV (65.05%，P＜0.01) were better, and SW had a certain direct killing effect on BVDV, but the blocking effect of SW on the invasion of BVDV was poor.【Conclusion】The effect of swainsonine (SW)-anti-BVDV activity in vitro was better, and the authors infered that the antiviral mechanism of SW might be that it could enter into the cells and inhibit the reproduction of BVDV, or it could play a role by killing the uncombined BVDV.

Sugar-Nitrogen Characteristics of Wheat at Yield Level of 9 000 kg•hm-2 Following Rice

DING Jin-Feng-1, 2 , ZI Yan-1, CHENG Ya-Mei-1, PAN Ting-1, FENG Chao-Nian-1, 2 , ZHU Xin-Kai-1, 2 , LI Chun-Yan-1, 2 , PENG Yong-Xin-1, 2 , GUO Wen-Shan-1, 2

Scientia Agricultura Sinica. 2014, 47(1): 182-190. doi:10.3864/j.issn.0578-1752.2014.01.019

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【Objective】 The sugar-nitrogen characteristics of wheat at yield level of 9 000 kg•hm-2 following rice were studied. In addition, the sugar-nitrogen nutrition diagnosis indexes for wheat at yield level of 9 000 kg•hm-2 were suggested. 【Method】 Wheat populations at two different yield levels in rice-wheat rotation, including ＜9 000 kg•hm-2 population (hereinafter as HY) and ≥9 000 kg•hm-2 population (hereinafter as SHY), were established by agronomic management of the medium-gluten wheat, Yangmai 20 (Triticum aestivum L.). From 2010 to 2012, field experiments were conducted as split-plot design at 16 combinations of two applications of nitrogen amount (210 kg•hm-2 and 262.5 kg•hm-2), two nitrogen applied ratios of basis﹕tillering﹕elongation﹕booting (3﹕1﹕3﹕3 and 5﹕1﹕2﹕2), and four nitrogen topdressing stages (flag leaf stage, booting stage, heading stage and anthesis). The dynamics of soluble sugar content, nitrogen content and soluble sugar to nitrogen ratio (hereinafter as C, N and C/N) in plant population of different yield levels were analyzed. And at different growth stages, the relationship between plant C, N and C/N and grain yield were analyzed, respectively. In addition, the sugar-nitrogen nutrition diagnosis indexes for wheat at yield level of 9 000 kg•hm-2 were suggested. 【Result】 As the developmental process goes on, the plants C and C/N had two peak values at the beginning of over-wintering stage and at stage from booting to anthesis, and a low value at green-turning stage. Then, the plants C and C/N showed a gradual decrease after anthesis. The plants N, however, showed a gradual decrease during the growth period - rapid decrease during the period between the beginning of over wintering stage and elongation stage, slow decrease during the period between elongation stage and booting stage, and slow decrease during the period between booting stage and maturity stage. In SHY plants, C and N at booting and N at anthesis were significantly higher than those of HY. But C at maturity was significantly lower than those of HY in 2010-2011, and not-significantly lower than those of HY in 2011-2012. In addition, C/N at maturity was significantly lower than those of HY. Plants C and N at booting stage and N at anthesis were in a positive linear correlation with grain yield. Plants C and N at milk-ripe stage were in a parabolic cure relationship with grain yield. Plants C at maturity were in a negative linear correlation with grain yield. The sugar-nitrogen nutrition diagnosis indexes for the population of about 9 000 kg•hm-2 were suggested. At the stages of booting, anthesis, milk-ripe and maturity, C were 14.56%-16.78%, 14.52%-16.82%, 10.59%-11.23% and 1.62%-1.76%, respectively, N were 1.55%-1.64%, 1.47%-1.57%, 1.28%-1.30% and 1.15%-1.20%, respectively, and C/N were 9.37-10.25, 9.80-10.69, 8.29-8.77 and 1.41-1.48, respectively. 【Conclusion】 Compared with HY, from the beginning of over wintering stage to elongation, C and N in SHY plants were generally high, and C/N was relatively suitable. From booting stage to anthesis, C and N in SHY plants were significantly higher. After anthesis, SHY plants could accumulate more sugar and had a higher conversion rate of sugar to grain yield as compared with HY.

Defense Enzyme Activity of Winter Jujube at Different Stages Induced by the Damage of Apolygus lucorum

LI Lin-Mao-1, 2 , MEN Xing-Yuan-1, YE Bao-Hua-2, YU Yi-1, ZHANG An-Sheng-1, LI Li-Li-1, ZHOU Xian-Hong-1, ZHUANG Qian-Ying-1

Scientia Agricultura Sinica. 2014, 47(1): 191-198. doi:10.3864/j.issn.0578-1752.2014.01.020

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【Objective】The objective of this study is to compare the activities of defense enzymes in different tissues of winter jujube induced by the damage of Apolygus lucorum and artificial needling, and to observe the changes of stress defense activities of enzymes in leaves, buds, flowers and young fruits with different levels of damage by A. Lucorum. 【Method】The experiments were conducted with vulnerable jujube tissues inoculation, artificial simulated needling, counting damage spots after 24 hours, then the damaged tissues were sampled. The activities of SOD, POD and CAT were determined by nitroblue tetrazolium method, guaiacol method and ultraviolet absorbance method, respectively.【Result】The activity of defense enzymes was changed with different damages. The activities of SOD, POD and CAT in leaves induced by the damage of A. lucorum and artificial needling were significantly higher than the leaves without damage, and the activities of SOD and CAT induced by A. lucorum damage were significantly higher than the artificial needling. There was not significant difference among the SOD activities in buds without damage, and those damaged by A. lucorum and artificial needling, while POD activities were significantly increased with the damage of A. lucorum and artificial needling. CAT activity in buds induced by A. lucorum damage was significantly higher than needling ones. At the flowering stage, SOD and CAT activities had no significant difference while POD was significantly increased with the damage stress, and that induced by A. lucorum damage was significantly higher than needling ones. At the young fruiting stage, the POD activities which were induced by A. lucorum damage and artificial needling were significantly higher than those without damage. At the same time, SOD activity was significantly higher in A. lucorum damaged fruit, while CAT activity was statistically indifferent. These results indicated that the defense enzymes in the tissues of winter jujube varied at the damage stress. Stress defense enzyme activities of winter jujube differed to varied damage levels of A. lucorum. With increasing damage, SOD activities in leaves and flowers increased initially and then decreased, while the activities of POD and CAT increased. With the increase of damage level, SOD activity in buds was statistically indifferent, while that of POD and CAT increased significantly. With the increasing damage, SOD activity of young fruits decreased initially, and then increased, however, that of POD increased initially and then decreased. 【Conclusion】The damage of A. lucorum could induce a series of biochemical reactions in host-plants. The activities of SOD, CAT and POD in leaves and the activity of POD in the reproductive organs (buds, flowers and fruits) were much higher under the damage stress. Both physical and chemical damages of A. lucorum induced more defense enzymes than the artificial needling. Defense enzymes in different tissues of winter jujube showed variations with the increasing levels of damage.

Effects of Ovariectomy on Serum GH Levels and Expression of GHR in Some Tissues of Female Goats

ZHANG Lei-1, WANG Yan-Yan-1, ZHOU Zhan-Qin-1, LI Guang-1, FU Ming-Zhe-1, ZHANG Suo-Liang-2, YIN Hai-Ke-2, ZHANG Sheng-Gang-2, REN Bao-Hua-2

Scientia Agricultura Sinica. 2014, 47(1): 199-208. doi:10.3864/j.issn.0578-1752.2014.01.021

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【Objective】This study was conducted to investigate the effects of ovariectomy on the meat performance, serum GH (growth hormone) levels and the relative expression levels of GHR (growth hormone receptor) mRNA in longissimus and biceps femoris tissues, liver and perirenal fat tissue, and to reveal the reasons of fattening after ovariectomy in female goats.【Method】Forty young hybrid does Kid of Boer Goats (Boer goat ♂ × Guanzhong dairy goat ♀) with similar body weight at about 5 months old were randomly divided into treatment group (n=20) and control group (n=20), the works of insect repellent and epidemic prevention were implemented a month ago before the beginning of the experiment. The goats in the treatment group were ovariectomized at the beginning of the experinment and the goats were not done in control group. Venous blood samples (10 ml) were collected from each goat to separate serum via jugular vein puncture using clean sterile syringes and needles with no additive at 8:00 in the morning on days 0, 10, 20, 30, 40 and 50 during the experiment. Then the serum was separated from blood samples to determine the levels of the growth hormone (GH) with specific ELISA kits (enzyme-linked immunosorbent assay kit) according to the manufacturer’s instruction. The slaughter rate, lean percentage of carcass, loin eye area and proportion of bone and meat were measured after 10 goats (5 goats of each group) were slaughtered on the 50 th day. At the same time, longissimus and biceps femoris tissues, liver and perirenal fat samples were collected immediately after goats were sacrificed, then three biological replicates were collected at each sample, and the samples were rinsed with sterile saline to remove blood clots and wrapped in aluminum foil with good marks, shock-frozen in liquid nitrogen. The mRNA expression levels of GHR gene in liver, longissimus muscle, biceps femoris and perirenal fat were detected with real-time quantitative PCR (qRT-PCR) and the method of 2-ΔΔCt (the Ct mean value of threshold cycle). 【Result】The serum GH levels of the ovariectomized goats on the 20th day showed an upward trend and were up to 9.50 μg•L-1 which were significantly higher than those of the controls (P＜0.05). On the 50th day, the levels were up to 11.55 μg•L-1 that was 2.21 μg•L-1 higher than those of the control group (9.34 μg•L-1) significantly (P＜0.01). In addition, the goats in treatment group with higher slaughter rate, lean percentage of carcass and loin eye area compared with that in the control group. The slaughter rate in ovariectomized goat was up to 45.62% which was higher than that in the control group (42.41%) and the difference was significant (P＜0.05), but the goats had a lower proportion of bone and meat (P＜0.05) in treatment group. In this study, the expression levels of GHR gene in liver of ovariectomized goats were 3.21 times (P＜0.01) more than that of the control group. Compared with the control group, a very significant increase in the GHR mRNA level was observed in longissimus of ovariectomized goats, and the expression level was the highest in the present study which was 4.87 times higher than that of the control group (P＜0.01). At the same time, a higher expression of GHR mRNA in biceps femoris (3.17 times) was also observed in ovariectomized goats, and the difference was significant compared with the control group (P＜0.05). As to perirenal fat, the expression levels of GHR gene in treatment group was 1.91 times more than that in the control group, and the difference was significant (P＜0.05)【Conclusion】 Results of the study showed that there were significant effects of ovariectomy on slaughter rate, proportion of bone and meat, serum GH levels. Meanwhile, the expression levels of GHR gene in liver, longissimus muscle, biceps femoris and perirenal fat tissues of female goats were also influenced by the ovariectomy. It is indicated that the ovariectomy increased the slaughter rate and decreased the proportion of bone and meat through enhancing the levels of serum GH and expression levels of GHR gene in the target tissues, and improved the meat performance of ovariectomized goats finally.

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