Table of Content

01 July 2016, Volume 49 Issue 13

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

QTL Mapping of Self-Compatibility, Silique and Seeds-Associated Traits in Brassica rapa

SUN Qian-qian, WU Jian, CHENG Feng, WANG Xiao-wu, LIANG Jian-li, SHEN Shu-xing

Scientia Agricultura Sinica. 2016, 49(13):  2449-2458.  doi:10.3864/j.issn.0578-1752.2016.13.001

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【Objective】Brassica rapa is an important oil crop in the world. Higher seed yield with good quality is one of the important agronomic traits. Due to the self-incompatibility characteristic of most B. rapa, it is difficult to purify breeding materials and realize maximum yield of this plant, since the seed yield of this crop is influenced by the silique related traits, such as the number of pods, number of seed per silique. Besides, the seed quality is closely related to seed color. The aim of this research is providing an important genetic basis for breeding self-compatibility varieties or inbred lines of B. rapa and genetic modification of yield with quality in this crop.【Method】The Recombinant Inbred Lines (RILs) population derived from the cross between genotypes R-O-18 with well self-compatibility and L58 with weak self-compatibility consisting of 117 lines was used to conduct QTL analysis. Based on previous linkage map generated by 372 InDel markers, the Interval Mapping (IM) method was used to survey a total of nine traits as follows: compatible index of flowering time (CIFT), pods rate (PR), silique length (SL), silique beak length (SBL), silique length/silique width (SL/SW), silique beak length/silique length (SBL/SL), number of seed per silique (NSPS), thousand seed weight (TSW) and seed color (SC).【Result】RILs population in the 9 self-compatibility, siliques and seeds-related traits showed continuous variation and variation margin, showed normal or partial normal distribution, with typical quantitative genetic characteristics. CIFT showed an extremely significant positive correlation with PR, SL, SBL, SL/SW, NSPS, and TSW, where the correlation coefficient between CIFT and NSPS was the largest, reaching 0.8487; and several other traits in order of size (high to low): TSW, PR, SBL, SL, and SL/SW. NSPS and TSW presents a very significant positive correlation with PR, SL, SBL, SL/SW, NSPS also had a significant positive correlation with TSW (0.6477). The PR, SL and SBL, SL/SW also showed a significant positive correlation. SBL/SL showed a negative correlation and a significant positive correlation with SBL, but not correlated with other traits. A total of 12 QTL loci were detected and the major ones were located on chromosome A09, and totally 10 QTLs explained phenotypic variation of more than 10%. Two QTLs controlling CIFT were located on chromosome A09, accounting for the phenotype variation 13.1% and 16.7%, respectively. Six QTLs related to silique were located on A06, A09 and A10, respectively, explaining phenotypic variance of 13.4%-17.7%. Furthermore, four seed-associated QTLs were located on chromosome A02, A05, A06 and A09, respectively, with QTL effects varied from 7.9% to 42.1%, of which the QTL located on A09 was the major QTL for SC, as it accounted for phenotypic variance of 42.1% and it had additive effect value of 1.22. 【Conclusion】A total of 12 QTLs which control self-compatibility (2), siliques (6) and seeds-related traits (4) were detected. For the two self-compatibility QTLs, they are different with the S locus identified previously, which indicated that the two loci detected in this study may be the non-major QTLs. Additionally, one QTL of the 4 seeds-related QTLs is the major QTL of this trait.

A Service Performance Evaluation System of National Crop Germplasm Resources Infrastructure

CHEN Li-na, FANG Wei, SI Hai-ping, CHEN Yan-qing, CAO Yong-sheng

Scientia Agricultura Sinica. 2016, 49(13):  2459-2468.  doi:10.3864/j.issn.0578-1752.2016.13.002

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【Objective】National crop germplasm infrastructure changed into full operational service stage during the 12th Five-Year Plan, it is necessary to construct an effective service performance evaluation system to evaluate the service effectiveness quantitatively, so that the management department can allocate funds according to how the service function was implemented, and ensure the infrastructure to work better and improve service effectiveness. 【Method】 On the basis of analyzing relevant factors that influence service effect, considering the reality of national crop germplasm infrastructure, this paper constructed a service performance evaluation system of multi-dimension and multi-index based on Analytic Hierarchy Process(AHP) , and it followed scientific, comprehensive, systematic and sustainable evaluation principles. Firstly, this paper referenced these service performance evaluation indexes drafted by National Science and Technology Infrastructure Center, classified and integrated them into different levels according to relevant factors, every level had different evaluation sub-indexes. Then the paper improved the classical AHP, calculated the index relative weight using expert scoring, averaging and normalizing method, and multiplied different level index weights to get actual weight. Finally, the paper weighted these index weights and their service numbers, so as to get overall evaluation value and part evaluation value.【Result】This paper analyzed the yearly service data of national crop germplasm infrastructure during the 12th Five-Year Plan quantitatively, the experimental results were as follows: (1) The overall service effectiveness was good and increased steadily during the 12th Five-Year Plan. (2) The integrated resources total increased continuously, but the growth rate was slow, and the resources number of evaluation and update was small. (3) The management service standardized orderly and had little change over the years. (4) The service effectiveness increased obviously, the amount of resources services and information services were large, while the amount of technology services was small. (5) The user group was relatively stable, and the proportion of serving company users was increasing. (6) The future service potential enhanced greatly. (7) The technology services and the typical services grew significantly, but the number was still limited. So, in the future, we should integrate more resources, accelerate evaluation and update of germplasm resources to meet the needs of different users, so as to improve service depth. We also should strengthen infrastructure, improve the management system, and make service personnel more professionalization to improve service efficiency. What’s more, we should still focus on serving company and individual users, enhance innovation capability to improve service breadth. Finally, we should attach importance to the role of high level scientific research brought by germplasm resources service, standardize subject service process to further improve service quality.【Conclusion】The service performance evaluation system of multi-dimension and multi-index can evaluate service effectiveness comprehensively, and the evaluation indexes embody service depth, effectiveness, breadth, quality and potential, etc.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Risk Assessment of Cold and Hot Damages for Double-Cropping Early Rice (DCER) in Lower-Middle Reaches of the Yangtze River Basin

WANG Chun-yi, YAO Peng-juan, ZHANG Ji-quan, REN Yi-fang

Scientia Agricultura Sinica. 2016, 49(13):  2469-2483.  doi:10.3864/j.issn.0578-1752.2016.13.003

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【Objective】Agricultural meteorological disasters are the important factors which threaten the national food security. Risk assessment on agro-meteorological disasters is the main research direction of guaranteeing agricultural production. There is the largest double-cropping early rice (DCER) planting area in Yangtze River basin in China, the DCER output in this area accounts for more than half of the country’s total production. This region is located in central China, where the hydrothermal resources are relatively abundant, but the seasonal distribution is uneven. Temperature swings in spring and hot days emerge frequently in summer, causing DCER’s cold and hot damages and affecting final yield. Therefore, it is emergency to carry out risk assessment of cold and hot damages of DCER. 【Method】The research was based on the meteorological data during 1961-2012, agricultural meteorological data during 1981-2010 and social statistics data of DCER growing region in lower-middle reaches of the Yangtze River Basin. First and foremost, four assessment models were built to evaluate cold and hot damage hazard, environmental vulnerability, exposure, disaster prevention and mitigation capacity. For laying a foundation for the formation mechanism of natural disaster risk, a risk evaluation model was developed in terms of hazard which is fine depicted in development phrase, vulnerability, exposure and disaster prevention and mitigation capacity. Weight coefficients of the four factors are calculated by entropy weight evaluation method. The results of the multi-risk assessment model is valuable for decision making to release disaster risks.【Result】 The hazard assessment model was constructed based on disaster intensities at various developmental stages, the weight coefficient of the development stage and the weight coefficient of cold and hot damages. The assessment results show that the planting area in southern Hunan and southeastern Jiangxi is suitable for DCER, where cold and hot damages are rarely happened. Hazard in center of Hunan and Jiangxi is 0.3, slightly higher due to hot damage at filling stage, while the higher hazard in eastern Hubei is caused by cold damage. Zhejiang is reduced to absolute inferior planting area for its higher hazard during the whole growing season except tillering stage, especially the serious hot damage at filling stage. Vulnerability assessment model was constructed of index of yield variation. The assessment results show that mid-east Zhejiang, mid-south Jiangxi and Hubei planting areas are the lowest vulnerable areas. Vulnerability in northeastern of Jiangxi is quite high, followed by Ningxiang and Chaling of Hunan province, where yield is quite sensible to weather condition. The exposure assessment model was constructed based on the index of vegetation coverage. The assessment results show that DCER planting area ratio is up to 85% in the eastern of Hunan and northeastern of Jiangxi, which means high exposure to the rice production. The exposure is lower in Zhejiang and Hubei, and it won’t have high impacts on DSER yield. The disaster prevention and mitigation capacity assessment model was constructed based on the total powers of agriculture machine, net income per capita and chemical fertilizers. The assessment results show that disaster prevention and mitigation capacity is highest in Zhejiang province. It is relatively higher in central Hunan, western Hubei and southern Jiangxi. Disaster prevention and mitigation capacity is lower in the other areas. The risk evaluation model was constructed based on hazard, vulnerability, exposure, disaster prevention and mitigation capacity. Mid-west of Zhejiang, northeast of Jiangxi, central Hunan and east of Hubei are divided into high-risk areas. Southern Hunan, southeastern Jiangxi, eastern Hubei and eastern Zhejiang are roughly divided into the low-risk and the other areas are divided into medium risk areas. 【Conclusion】 Diverse measures should be adopted to lesson the risk of DCER planting in each province in Yangtze River basin: in mid-west of Zhejiang, it is useful to adjust the sowing period. For northeastern Jiangxi, increasing the investment of local agriculture is needed. It is necessary to adjust the planting structure in Hunan. Science and technical inputs in Hubei should be increased to reduce the risk.

Influence of Returning Corn Stalks to Field Under Different Soil Moisture Contents on Root Growth and Water Use Efficiency of Wheat (Triticum aestivum L.)

ZHANG Su-yu, WANG He-zhou, YANG Ming-da, WANG Jing-li, HE De-xian

Scientia Agricultura Sinica. 2016, 49(13):  2484-2496.  doi:10.3864/j.issn.0578-1752.2016.13.004

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【Objective】To evaluate the influence of soil moisture and corn stalks returning practice on wheat root growth, grain yield and water use efficiency, a 2-year micro-plot experiment was conducted under rain-proof shelter condition, aiming at provide reference for improving effect and application of stalks returning to field. 【Method】Prior to wheat sowing, corn stalks were cut and incorporated into soil by plowing (stalks returning, RS), with non-stalks returning as control (CK). Both RS and CK were subjected to 50%-55% (Drought, D), 60%-65% (Slight Drought, SD) and 70%-75% (Normal, N) of field moisture capacity, respectively.【Result】The results showed that nodal roots per plant at maturity, root activity, root dry weight density within 0-25 cm soil layer of winter wheat in D treatments at different growing stages were dramatically decreased compared with those in either SD or N treatments, grain yield was decreased by 4.34%-38.30% and 14.30%-36.63%, respectively, though consumption of soil water storage and water use efficiency in D treatments increased by 7.92%-25.56% and 31.34%-90.72%, 12.69%-30.09% and 11.83%-32.88%, respectively. In D treatment, nodal roots per plant were 17.17%-29.41% and 5.60%-27.86% higher than those in CK at re-growing and maturity stages, but root dry weight density within 0-25 cm soil layer at different growing stages was lower in RS treatments than that in CK. The decrement in both root vigor and root dry weight density within 25-50 cm soil layer after flowering was significant higher than those in CK, grain yield and water use efficiency in RS were significantly decreased by 15.02%-19.52% and 7.51%-14.56% compared with that in CK. In SD and N treatment, nodal roots per plant were higher at different growing stages and the decrement in both root vigor and root dry weight density within 25-50 cm soil layer were lower in RS treatments, while irrigation amount and total water consumption were all decreased. Consumption of soil water storage was significantly increased, except for the N treatment in 2013-2014, grain yield and water use efficiency were dramatically increased by 6.09%-9.18% and 6.77%-11.13% compared with those in CK treatments. In addition, the present study also showed a significant interaction both of grain yield and water use efficiency between corn stalks returning and soil moisture conditions.【Conclusion】Corn stalks returning practice reduced irrigation amount, improved grain yield and water use efficiency by conserving soil water storage and improving root physiological function during the whole growing period under normal soil water and even slight drought conditions, but decreased grain yield and water use efficiency under drought condition.

Cotton Chemical Topping with Mepiquat Chloride Application in the North of Yellow River Valley of China

LI Fang, WANG Xi, WANG Xiang-ru, DU Ming-wei, ZHOU Chun-jiang, YIN Xiao-fang, XU Dong-yong, LU Huai-yu, TIAN Xiao-li, LI Zhao-hu

Scientia Agricultura Sinica. 2016, 49(13):  2497-2510.  doi:10.3864/j.issn.0578-1752.2016.13.005

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【Objective】The objective of this study is to investigate the feasibility of cotton chemical topping with mepiquat chloride (1,1-dimethyl-piperidinium chloride, DPC) application in the North of Yellow River Valley of China. 【Method】This study was conducted with GX3, XK4, S126 and XS17 as materials in Hejian city in Hebei province and at Shangzhuang Experimental Station of China Agricultural University in Beijing from 2012 to 2014, consisting of six independent experiments. The chemical topping with DPC application included conventional DPC (98% soluble powder, provided by Jiangsu Runze Agrochemical Co. Ltd.) application alone and the combination of conventional DPC application with fortified DPC (DPC⁺, 25% slow-released emulsion, oil in water, developed by Beijing Agricultural Technology Extension Station and Engineering Research Center of Plant Growth Regulator of China Agricultural University, and provided by Xinjiang Golden Cotton Technology Co., Ltd.) application during flowering and fruiting period. These two strategies were hereafter referred to as DPC and DPC+DPC⁺, respectively. The manual topping accompanied with conventional DPC application was served as control (hereafter referred to as DPC+MT). 【Result】 In 2012 and 2013, characterized by great precipitation during flowering and fruiting period (from July to August), DPC and DPC+DPC+ produced significantly 10.6-12.3 cm higher plant and 5.8-7.9 more sympodials compared with DPC+MT. In 2014, characterized by drought in summer, there were no significant differences in plant height between DPC or DPC+DPC⁺ and DPC+MT, and the number of sympodials of chemical topping only increased by 0-3 relative to control. DPC and DPC+DPC⁺ did not significantly affect cotton yield in all experiments, but a reduced yield of DPC+DPC⁺ was noticed in 2012, companied with the less bolls on the upper sympodials but more bolls on the new sympodials occurred after DPC⁺ application as well as a delay of maturation. In terms of 2013 and 2014, DPC+DPC⁺ showed similar yield and maturation to those of DPC+MT. The application time (from mid-July to the end of July) and doses (varying from 750 to 1 500 mL·hm^-2) of DPC⁺ had no significant effects on both plant architecture and cotton yield. While in case of extending the maturity of late bolls, it should be avoided to use large doses of DPC⁺ at the end of July. Compared with DPC+DPC⁺, the conventional DPC application alone (DPC) showed a weaker control in terms of plant height and the number of sympodials, and possibly had a risk of reduced yield in rainy years or at higher plant density. 【Conclusion】Cotton chemical topping with the application of DPC or DPC+DPC⁺ is basically feasible in the North of Yellow River valley of China. And while make a decision whether applying conventional DPC alone or applying DPC+DPC⁺, the meteorological factor, especially precipitation and plant density should be considered carefully.

PLANT PROTECTION

Guidance: Effective Management of Bemisia tabaci Relies on Fundamental Research

YAN Feng-ming

Scientia Agricultura Sinica. 2016, 49(13):  2511-2513.  doi:10.3864/j.issn.0578-1752.2016.13.006

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Differential Physiological and Biochemical Responses of Cucumber to the Feeding by Bemisia tabaci B and Q Biotypes

LIU Ming-yang, LEI Cai-yan, LI Jing-jing, LU Shao-hua, BAI Run-e, TANG Qing-bo, YAN Feng-ming

Scientia Agricultura Sinica. 2016, 49(13):  2514-2523.  doi:10.3864/j.issn.0578-1752.2016.13.007

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【Objective】B (Middle East-Asia Minor 1) and Q (Mediterranean) biotypes, two cryptic species of Bemisia tabaci species complex, are important agricultural pests. Extensive applications of insecticides for control of the pests have resulted in pesticide resistance in the whiteflies, have endangered ecological safety and human health. Implementation of safe pest management strategy is therefore becoming very important and urgent. Utilization of plant defense is one of the important components in integrated pest management. The objective of this study is to investigate the differential responses of nutrients and defensive enzymes in cucumber induced by the feeding of B. tabaci B and Q biotypes and thereby to elucidate the physiological and biochemical mechanism underlying defence responses of cucumber to B. tabaci.【Method】Adults of B. tabaci B and Q biotypes and cucumber plants (var. Bojie-I) from the laboratory cultures were used for the experiments. The cucumber plants at four-leaf stage were respectively used to feed 200 adults of B. tabaci B and Q biotypes, with healthy plants as controls, and the contents of nutrients (soluble sugar and soluble protein) and polyphenolic contents, activity of phenylalanine ammonialyase (PAL), the enzyme of defensive substance biosynthesis, and activity of protective enzymes in cucumber were determined after continuously feeding for 1, 3, 5, 7 and 9 days by B and Q biotypes of B. tabaci. 【Result】Within the experimental period, the contents of soluble sugar and protein in cucumber increased after 3 d by B. tabaci B biotype, but decreased after 1 d by B. tabaci Q biotype, compared to those in controls. The contents of polyphenols and activity of PAL, the key enzyme in its biosynthesis pathway, increased after infested both by B and Q biotypes, with higher activities to Q biotype feeding. The activity of superoxide dismutase (SOD) and catalase (CAT) increased and the activity of peroxidase (POD) decreased after feeding by B biotype, while the activities of SOD increased and the activity of POD and CAT decreased after feeding by Q biotype. 【Conclusion】Differential changes of nutrients and defensive enzymes in cucumber after the feeding of B. tabaci B and Q biotypes were found in the present study. Both B and Q biotypes of B. tabaci were able to induce the synthesis of defensive substance polyphenols, and increased the content of polyphenols in cucumber; but there were differences in inducing the contents of nutrients and the activities of protective enzymes, i.e., more nutrients in cucumber by B biotype feeding, while higher activities of defensive enzymes by Q biotype feeding. Those different changes in induced physiological and biochemical responses in plants to the herbivore may result from the difference in host plant adaptability between B and Q biotypes of B. tabaci. These results provide a basis for pest management strategies, especially for utilization of plant defense as the main control tactic, so as to target different biotypes of B. tabaci on different host plants.

Analysis of Physiological Characteristics with Response to Bemisia tabaci B Biotype in Different Resistant Varieties of Tomato

CAI Chong, XU Ying-ying, CUI Xu-hong

Scientia Agricultura Sinica. 2016, 49(13):  2524-2533.  doi:10.3864/j.issn.0578-1752.2016.13.008

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【Objective】The objective of this study is to clarify the physiological characteristic changes inside the leaves of tomato between the resistance and the susceptible cultivars under the stresses of Bemisia tabaci B biotype, and to provide a reference for the breeding and popularization of tomato varieties resistant to B. tabaci.【Method】 An experiment was carried out under artificial climate conditions. The resistant variety (Hongshengnv, HSN) and the susceptible variety (Huangshengguo, HSG) were infested by B. tabaci B biotype in a no-choice way. The contents variation of the secondary metabolic materials (i.e., sucrose esters, gallic acid, caffeic acid, ferulic acid, benzoic acid, salicylic acid, lignin), the changes of some protective enzymes activities (i.e., POD, CAT, PPO, LOX) and trypsin proteinase inhibitor activity (TI), the value variation of two photosynthetic parameters (i.e., Fv/Fm, Fv/Fo), the changes of cell membrane lipid peroxidation (i.e. H₂O₂ content, production rate, MDA content and electric conductivity (EC)), were determined all together at the same time.【Result】There were marked differences in the contents of the SE, phenolic acids, lignin, H₂O₂, and MDA between the HSN and the HSG. Meanwhile, the differences of the activities of TI and protective enzymes, the values of Fv/Fo, were also significant between these two varieties. However, the value differences of these parameters, such as Fv/Fm, production rate, and EC, were not obvious. The two varieties showed different responses to the infestation of B. tabaci B biotype. For example, after 9 h, the contents of sucrose esters, phenolic acids, and lignin as well as the activities of protective enzymes increased all together. However, there were considerable raises as to its extent with comparing HSN to the HSG. Photosynthesis was inhibited in the leaves, confirmed by the observation of reducing of the activities of TI and photosynthetic parameter values. There were reducing to lower intensities with comparing the HSN to the HSG. More active oxygen, MDA and EC found in the latter indicated that HSG cell had been damaged greatly.【Conclusion】There were different responses to the stresses of B. tabaci B biotype as to tomato of different resistant varieties. Higher variation of secondary metabolic materials contents and protective enzymes activities were observed in the resistant variety. Meanwhile, the photosynthetic parameter values, active oxygen content, cell membrane lipid peroxidation, as well as the TI activities changed more dramatically in the susceptible variety.

Identification and Expression Analysis on Lysozyme Gene of Bemisia tabaci

YU Jie, WANG Deng-jie, LEI Zhong-ren, WANG Hai-hong

Scientia Agricultura Sinica. 2016, 49(13):  2534-2543.  doi:10.3864/j.issn.0578-1752.2016.13.009

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【Objective】The objective of this study is to provide a basis for understanding the important role of lysozyme genes in Bemisia tabaci infected by the fungus Beauveria bassiana , identify lysozyme genes and analyze their sequence features, evolutionary relationships and expression pattern, and to provide a theoretical foundation for charifying innate immunity in B. tabaci. 【Method】Target genes were screened from the results of the second-generation high-throughput sequencing against the redundancy nucleotide databases when the E value ＜10^-5. The open reading frame and amino acid sequence of BtLyzs were found using the ORF Finder software; the protein domains of BtLyzs were predicted using Pfam and SMART software; the signal peptide sequence of BtLyzs was predicted using SignalP 4.1 Server; the amino acid sequence alignment of BtLyzs was performed using the MEGA6.0 software; BtLyzs were characterized by using phylogenetic analysis with homologous genes of 31 other insects, a neighbor-joining of phylogenetic tree was constructed using the MEGA 6.0 software for further analysis and identification of the genes. The expression pattern of screened genes were determined at egg, nymph and adult stages of B. tabaci which were infected by the fungus Be. bassiana after 12, 24, 36, 48, 60 h using the quantitative real-time PCR (qRT-PCR). 【Result】 Four lysozyme genes were identified and designated as BtLyz1, BtLyz2, BtLyz3 and BtLyz4, which were 1 819, 1 149, 829, 928 nt, respectively. They were predicted to encode proteins of 159, 160, 148, 160 amino acids, respectively. Amino acid sequence alignment, phylogenetic analysis and protein tertiary structure prediction showed that BtLyz1 and BtLyz4 belong to i-type lysozymes, BtLyz2 and BtLyz3 belong to c-type lysozymes. BtLyz-1 formed a clade specific with Acyrthosiphon pisum of ApLyz-i1, BtLyz4 formed a clade specific with Diaphorina citri of DcLyz-i3 and Acyrthosiphon pisum of ApLyz-i2; BtLyz2 and BtLyz3 formed a clade specific with Nilaparvata lugens of NlLyz-c1 and Harmonia axyridis of HaLyz-c3. Compared to the control, the relative expression of all four genes in egg and BtLyz4 in nymph were not induced significantly by fungi-infected; the transcription of BtLyz1 nymph stage underwent the fluctuation of up-regulation, down-regulation, and second up-regulation and peaked at 24 h, it was increased 4.55 folds compared to the control; the transcription of BtLyz1 and BtLyz4 adult stage underwent the fluctuation of up-regulation, down-regulation, and second up-regulation and peaked at 60 h, they were increased 11.31 and 4.21 folds compared to the control. The transcription of BtLyz2 and BtLyz3 nymph stage underwent the fluctuation of up-regulation and down-regulation and peaked at 24 h, they were increased 5.09 and 8.31 folds compared to the control, then down-regulated obviously at 60 h, they were reduced 0.19 and 0.13 folds compared to the control. The transcription of BtLyz2 and BtLyz3 adult stage underwent the fluctuation of up-regulation and down-regulation and peaked at 24 h, they were increased 5.56 and 8.84 folds compared to the control. 【Conclusion】 Four BtLyz genes were identified in B. tabaci. Among them, two are c-type and two are i-type lysozymes. Transcriptional levels of four BtLyzs genes in B. tabaci were induced through different developmental stages and different time points in fungi-treated individuals compared to the control, they were not induced significantly in eggs, and showed different expression trends in nymphs and adults. The four BtLyzs genes probably might participated in the innate immune responses to fungus infection, and could be a new potential target for biocontrol of B. tabaci.

Comparison of Feeding Behavior Between Two Bemisia tabaci Strains Using EPG Technique

ZHANG Wen-ping, LIU Bai-ming, ZHANG Shan, WAN Fang-hao, CHU Dong

Scientia Agricultura Sinica. 2016, 49(13):  2544-2552.  doi:10.3864/j.issn.0578-1752.2016.13.010

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【Objective】Cardinium is one of the endosymbiont species infects in sweetpotato whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). Previous studies have shown that there was a significant difference in the fitness between Cardinium- infected (C⁺) and -uninfected (C^-) strains of B. tabaci Q biotype. However, the behavioral mechanism causing the difference in fitness between the two strains is not clear. This study investigated the difference in the feeding behavior of these two strains to reveal the underlying mechanism responsible for the difference in fitness. 【Method】The feeding behavior of C⁺ strain and C^- strain on cotton and tomato during 6 h was recorded using an electrical penetration graph (EPG). The waveform types of feeding behavior were then identified and analyzed. A total of 19 parameters (11 parameters associated with non-phloem phase and 8 parameters associated with phloem phase) were calculated and analyzed to compare the two strains’ feeding behavior at phloem stage and non-phloem stage.【Result】Of the 118 successful recordings obtained in this experiment, 58 recordings (C^- strain=28 replicates and C⁺ strain=30 replicates) were on cottons and 60 recordings (C^- strain=31 replicates and C⁺ strain=29 replicates) were on tomatoes. The results showed that at the non-phloem phase on cotton C⁺ strain had a significantly longer total duration of probes than C^- strain. C+ strain had a significantly shorter time than C^- strain in terms of in duration of np after the first E. The parameters associated with salivation into a sieve element and ingestion of a sieve element sap had no significant difference between the two strains on cotton at phloem phase, and the percentage of whiteflies reaching phloem phase within 6 hours had no significant difference. Meanwhile, at the non-phloem phase, C⁺ strain had a greater number of total probes and probes before the 1st E than C^- strain on tomato. C⁺ strain also had a longer time from the 1st probe to the 1st sustained E2 and a shorter average probe time than C^- strain. The parameters regarding salivation into a sieve element and ingestion of sieve element sap also had no significant difference between the two strains on tomato at phloem phase. Also, the percentage of whiteflies reaching phloem phase within 6 hours had no significant difference. On the whole, the parameters associated phloem phase had no significant difference between the two strains. Compared with C^- strain, C⁺ strain has a longer probing time and requires more probes at non-phloem phase.【Conclusion】The feeding behavior of C⁺ strain and C^- strain has no significant difference at phloem phase, but does have a significant difference at non-phloem phase. The results indicate that the feeding behavior difference of the two strains at non-phloem phase is most likely related to the difference in fitness.

Integration of Emulsifiable Formulation Metarhizium flavoviride with Low-Rate Abamectin for Control of Bemisia tabaci Q Biotype

LI Mao-ye, CHEN De-xin, LIN Hua-feng, LI Shi-guang, PAN Jing, WU Sheng-yong

Scientia Agricultura Sinica. 2016, 49(13):  2553-2560.  doi:10.3864/j.issn.0578-1752.2016.13.011

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【Objective】 Bemisia tabaci, an important invasion insect pest in agriculture, has displayed a high resistance to many conventional pesticides. The objective of this study is to evaluate the synergistic effect of joint application of entomopathogenic fungi and chemical pesticides against B. tabaci in laboratory and field, and to provide a new approach for effective control of B. tabaci. 【Method】 Based on the previous study that a highly virulent of Metarhizium flavoviride strain (Mf96) was screened for control of B. tabaci Q biotype, three gradient emulsifiable formulation of Mf96 (1.0×10⁸, 1.0×10⁷, 1.0×10⁶ conidia/mL), which were combined with five low doses of abamectin EC (0, 15, 30, 45 and 60 µg·mL^-1) were separately sprayed to the body surface of 2nd instar nymphs of B. tabaci Q biotype in laboratory, and the mortalities were tested. The number of conidia deposited on the unit surface was counted under stereomicroscope. Single agent and their equal volume mixture of the suspension emulsion of Mf96 strain (1.0×10⁸ conidia/mL) and 1.8% abamectin WP (60 µg·mL^-1) were separately sprayed to control B. tabaci Q biotype on tobacco in the field, and the control efficacies were evaluated. 【Result】 LC₅₀ of Mf96 against 2nd instar nymphs of B. tabaci Q biotype decreased from 1 376 to 183 conidia/mm² during 4th to 8th day. After 7 days, LC₅₀ of the fungus combined with abamectin (60 µg·mL^-1) decreased from 378 to 46 conidia/mm². Abamectin had no significant effect on the fungal outgrowths and infection to 2nd instar nymphs of B. tabaci Q biotype at the low spray rates. The mixture suspension of different concentrations of fungus (low, medial and high) with abamectin (15, 30, 45 and 60 µg·mL^-1) resulted in different rates of mycotized cadavers, and high concentration of fungal mixture suspension with 30 µg·mL^-1 of abamectin caused 86.8% mortality of B. tabaci. No cadavers were observed in untreated control and single abamectin spray. Fungal conidia suspension emulsion, abamectin and their mixture were separately sprayed in the field, and the decline rates in nymphae of B. tabaci Q biotype population was the highest (53.6% and 85.7%) when the mixture was sprayed after 5 d and 10 d. Meanwhile, the changing trends of corrected control efficacies and population decline rate of B. tabaci Q biotype were similar at 5 random checks, the corrected control efficacy of the mixture was the highest (88.9%) after 25 d. The population decline rate of B. tabaci Q biotype in untreated control was negative value.【Conclusion】The combined of M. flavoviride with abamectin showed synergistic interaction, with additive effects on Q biotype B. tabaci. Therefore, it is an effective approach to control B. tabaci by combining M. flavoviride with abamectin at low spray rate.

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

Effect of Nitrogen Application Rates in Different Fertility Soils on Soil N Transformations and N Use Efficiency Under Different Fertilization Managements

YANG Xin-yi, LIU Xiao-hu, HAN Xiao-ri

Scientia Agricultura Sinica. 2016, 49(13):  2561-2571.  doi:10.3864/j.issn.0578-1752.2016.13.012

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【Objective】 The objective of this study is to research the effect of different nitrogen (N) application rates on soil labile N pools transformations (soil mineral N-SMN; soil soluble N-SSON; soil microbial biomass N-SMBN) and N use efficiency in soils after application of N fertilizer at wheat (Triticum aestivum L.) booting stage. 【Method】 A pot experiment with ¹⁵N isotopic tracer technique was conducted to study the soil labile N pools and the effect of supply N in different fertilization managements for 37 years (poor soil-NF: no application of fertilizer; low fertility soil-LF: inorganic fertilizer; moderate fertility soil-MF: low rate of organic fertilizer with inorganic fertilizer; high fertility soil-HF: high rate of organic fertilizer with inorganic fertilizer) after application of three different application rates of N (N0: 0, N1: 135 kg·hm^-2, N2: 180 kg·hm^-2) in soil and their relationships. 【Result】 SMN and SSON were the highest in the N1 treatment and then decreased with the application rate of N, but SMBN performed opposite tendency with the application rate of N, they were firstly decreased and then increased, and the highest in the N2 treatment. In the same application rate of N, SMN and SSON generally decreased in the order: high fertility soil＞moderate fertility soil＞low fertility soil＞poor soil, while SMBN generally decreased in the order: high fertility soil＞moderate fertility soil＞poor soil＞low fertility soil (P＜0.05). The increased amplitude of SMN, SSON and SMBN after addition of N into soils with different fertilities were the highest in the low fertility soil treatments, and were the lowest in the high fertility soil treatments. The soil N supply, NUE, N uptake by wheat and assimilated 15N-labeled fertilizer generally decreased in the order: high fertility soil＞moderate fertility soil＞low fertility soil＞poor soil (P＜0.05), respectively. The percentage of N from ammonium sulfate fertilizer by wheat to total N uptake by wheat generally decreased in the order: low fertility soil＞moderate fertility soil＞high fertility soil＞poor soil (P＜0.05). In the same soil fertility, the soil N supply, NUE, N uptake by wheat and assimilated ¹⁵N-labeled fertilizer were firstly decreased and then increased with the application rate of N, and were the highest in the N1 treatment (P＜0.05), as a whole, N from ammonium sulfate fertilizer by wheat/total N uptake ratio averaged 44%; meanwhile, the loss of 15N-labeled fertilizer generally decreased in the order: poor soil＞low fertility soil＞moderate fertility soil＞high fertility soil (P＜0.05). Furthermore, significant positive relationships were found between soil labile N pools and the soil N supply, NUE, N uptake by wheat and assimilated ¹⁵N-labeled fertilizer (P＜0.05). 【Conclusion】 In this experiment, appropriate application rate of N fertilizer (N3, 135 kg·hm^-2) in high fertility soil is beneficial to soil labile N pools transformations and has a high ability to synchronize the relationship between soil N supply and N requirements of crops, and increased the NUE, decreased the loss of fertilizer, so it may be an effective strategy for maintaining long-term soil fertility.

Effects of Mulching During Fallow Period on Soil Water Storage and Consumption and Its Relationship with Wheat Yield of Dryland

CHEN Meng-nan, SUN Min, GAO Zhi-qiang, REN Ai-xia, YANG Zhen-ping, HAO Xing-yu

Scientia Agricultura Sinica. 2016, 49(13):  2572-2582.  doi:10.3864/j.issn.0578-1752.2016.13.013

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【Objective】Strategies and practices are required to increase yield of dryland wheat and improve water use efficiency. The objective of this study was to confirm the effects of mulching during fallow period on the yield of dryland wheat, and to provide a solution to soil water retention.【Method】Field experiments were carried out with a winter wheat cultivar Yunhan 20410 from 2010 to 2013 in Qiujialing Village, Wenxi, Shanxi, and soil water storage was observed during fallow period and different wheat growth periods, yield and yield components were observed in two treatments, with and without mulching during fallow period after deep plowing. Changes in soil water storage and consumption, wheat yield and water use efficiency were analyzed.【Result】The results showed that soil water storage in the 0-3 m depth was increased at sowing stage by 47 mm in humid year under mulching during fallow period, 55 mm in normal year and 63 mm in dry year, and more water was stored in deep soil layers in dry year. Soil water storage efficiency was increased significantly, which was increased by 35% in humid year under mulching during fallow period, 48% in normal year and 101% in dry year, and its effect was still significant on the anthesis. The effects of water consumption during different growth periods were different under mulching during fallow period, which was increased significantly in the wheat growth period, but reduced significantly during fallow period, thus the annual water consumption did not change much. While the percentage of water consumption before jointing stage was decreased significantly under mulching during fallow period, water consumption and daily water consumption after jointing stage were increased significantly. The percentage of water consumption after jointing stage was also increased, especially the regulatory effect of water consumption at later growth stage of wheat in dry year. Wheat yield and yield components were increased significantly under mulching during fallow period, especially spike number, and wheat yield was increased by 30% in humid year, 35% in normal year and 50% in humid year. In dry year, spike number was increased by 19%, grain number per spike and 1000-grain weight also received a large effect. In addition, water consumption at each growth stage had close correlation with wheat yield under mulching during fallow period, especially after jointing stage. In mulching during fallow period treatment, soil water storage increased by 1 mm at sowing stage could improve wheat yield by 17-26 kg·hm^-2, soil water consumption in growth period increased by 1 mm could improve wheat yield by 22-26 kg·hm^-2. Rainfall productive efficiency and water use efficiency were increased significantly under mulching during fallow period, especially in dry year.【Conclusion】It could benefit the soil water storage from the fallow period to anthesis under mulching during fallow period. The annual precipitation was regulated by reducing water consumption at earlier growth stage, and increasing water consumption at middle and later growth stage under mulching during fallow period. In addition, the yield components were optimized, especially the spike, through the method, which was favorable for increasing production, and ultimately to achieve efficient use of rainfall. The effect of mulching during fallow period was better in dry year.

HORTICULTURE

The Relationship Between Sap Flow Velocity and Environmental Factors of the 19 a Apple Trees on the Loess Plateau at Different Time Scales

ZHANG Jing, WANG Li, HAN Xue, ZHANG Lin-sen

Scientia Agricultura Sinica. 2016, 49(13):  2583-2592.  doi:10.3864/j.issn.0578-1752.2016.13.014

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【Objective】The Changwu Tableland was taken as a study area in consideration of its typical representative of the Loess Plateau. The purposes of the study were to estimate water consumption rate of 19 a apple trees at different time scales by environmental factors, and provide a theoretical basis for the scientific distribution, sustainable management and production capacity of apple trees. 【Method】Thermal dissipation probe (TDP) was used to measure sap flow velocity of 8 standard 19 a apple trees on the Changwu Tableland located on the Loess Plateau from May to September, 2014, the data collector CR1000 (Campbell Scientific, UN) was used to collect and analyze the data, and the meteorological data were monitored continuously by automatic weather station 50 m away from the plot. The characteristics of sap flow were analyzed, and the relationship model between the rate of sap flow rate and environmental factors at different time scales was established.【Result】 The most close correlations between the sap flow velocity of the 19 a apple tree and various environmental factors at the hour scale was vapor pressure difference (VPD). Radiation (Rs), wind velocity (Ws) and soil temperature (Ts) also showed significant correlations with the sap flow. The sap flow curve had a single peak. The empirical model for the hour scale was: v=-11.683+2.3VPD+0.009Rs+0.55Ts+0.880Ws. At the day scale, Ts and VPD were significantly correlated with the sap flow velocity. The empirical model for the day scale was: v=1.637+0.404Ts- 3.097VPD. The sap flow velocity showed an order of August＞July＞June＞September＞May. Only Ts showed a correlation with the sap flow velocity. The empirical model for the month scale was: v=-3.524+0.509Ts. With the increasing time scales, the number of environmental factors associated with the sap flow velocity was gradually reduced, but Ts was the dominant factor at every scale and the correlation coefficients were gradually increased. Wind velocity (Ws) showed a significant correlation at the hour scale. VPD had a significantly positive correlation with the sap flow velocity at the hour scale and a significant and negative correlation at the day scale. The correlation between the Rs and sap flow velocity at the three time scales was not found. For the three stepwise regression equations, the correlation coefficient for the month scale was the largest. 【Conclusion】 At the larger time scale, the consumption of water can be estimated by monitoring a small number of environmental factors, but at the smaller time scales, most of the environmental factors will influence the sap flow. Therefore, estimates of the consumption by the environmental factors cannot be achieved with a great accuracy, and the method of TDP needs to be used to monitor the consumption.

Isolation of Chlorophyll Metabolism Key Genes and Molecular Mechanism of Green Fade in Guzmania Bracts Discoloration Process

LIU Jian-xin, DING Hua-qiao, TIAN Dan-qing, WANG Wei-yong, LIU Hui-chun

Scientia Agricultura Sinica. 2016, 49(13):  2593-2602.  doi:10.3864/j.issn.0578-1752.2016.13.015

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【Objective】 Guzmania is a trendy flower. Its flower formation and bracts discoloration process are often accompanied by green fade. To analyze the reason which bract’s green fade, it is necessary to isolate key genes and explore molecular mechanism of chlorophyll biosynthesis and degradation metabolism in the process of bracts discoloration. 【Method】 By cDNA library construction and EST sequencing batch, two key enzyme genes in chlorophyll biosynthesis pathway, including glutamyl-tRNA synthetase (GTS) and Uroporphyrinogen-Ⅲ synthase (UROS), were obtained. By homology cloning technology, a key enzyme gene in chlorophyll degradation pathway, pheophytin pheophorbide hydrolase gene (PPH), was obtained. Through measuring chlorophyll content using light transmittance measurement method, flavonoid content using HPLC method and analyzing gene expression pattern of key genes in chlorophyll metabolism using real-time quantitative PCR method, the molecular mechanism which green fade in discoloration bracts was studied. 【Result】 GTS obtained has 1 140 bp (GenBank: KP144289) in cDNA sequence, which compiles a 171 amino acid protein sequence, and has GlnRS-cataytic core and Nt-trans superfamily conservative regions; UROS obtained has 613 bp (GenBank: KP144288) in cDNA sequence, which compiles a 188 amino acid protein sequence, and has HemD and HemD Superfamily conservative regions; PPH obtained has 266 bp (GenBank: KP723523) in cDNA sequence, which compiles a 88 amino acid protein sequence, and has Abhydrolase-6 conservative region. Through measuring chlorophyll, flavonoid content and studying the expression level of key genes in chlorophyll metabolism, it could know that bract discoloration process accompanied by significant reduction of chlorophyll content and significant increase of flavonoid content. Furthermore, the expression level of chlorophyll biosynthesis key enzyme’s encoding genes, GTS and UROS, were also decreased obviously. As for the chlorophyll degradation key enzyme’s encoding gene, PPH, its expression level increased significantly at the beginning of bracts discoloration, then reduced to close to the lowest level, which in accord with green leaves, after finishing of discoloration. As for the control material, green leaf had the highest chlorophyll content and the lowest flavonoid content. Furthermore, the expression amount of chlorophyll biosynthesis related genes were the highest, while that of degrade related genes was the lowest. This was consistent with colorful plants in chlorophyll and flavonoid pigment change trend. 【Conclusion】 We obtained biosynthesis key enzymes genes, GTS, UROS, and degradation key enzyme gene, PPH, in chlorophyll metabolism pathway from Guzmania. The reason that bracts’ green fade was due to reduction of chlorophyll content, and accompanied by increase of flavonoid content. The reduction of chlorophyll content was due to chlorophyll’s biosynthesis reduced and degradation increased significantly. PPH played a key role in chlorophyll degradation process. The results will provide a basis for researching pigment change of view bract plants.

STORAGE·FRESH-KEEPING·PROCESSING

Gel Model and Mechanism of Low-Methoxyl Apple Pectin for Various pH

HAN Wan-you, DONG Gui-ru, QU Yu-ling MENG Yong-hong, GUO Yu-rong, DENG Hong

Scientia Agricultura Sinica. 2016, 49(13):  2603-2611.  doi:10.3864/j.issn.0578-1752.2016.13.016

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【Objective】The mathematical model of LM apple pectin gel strength which were prepared by citric acid - disodium hydrogen phosphate buffer solution were established with pH 1.78-6.62 and the mechanism of gelling was analyzed by data of rheology. The results of this study will provide a theoretical reference for the application of LMP in different pH environments. 【Method】Gel strength, rupture strength and rheological properties were measured by texture analyzer and rheometer, respectively. A model was established with Mate lab 10.0. According to the sol-gel transition point in graphs which was combined with storage shear modulus and loss shear modulus intersections in the graph, the pH range of gels could be determined. The mechanism of gels under different pH was analyzed on the basis of changes in storage shear modulus and loss shear modulus in the graph and the structure characteristics.【Result】The model of gel strength was followed by the polynomial law. In the range of pH 1.78-3.10, hydrophobic interactions between methoxyl ester groups and hydrogen bonds among undissociated carboxyl groups were formed which resulted in the sol to gel transition and gel strength to 21.19 g. When the pH of pectin solution was about 3.10, hydrophobic interactions between methoxyl ester groups and hydrogen bonds between undissociated carboxyl groups were formed. So that the elastic modulus increased significantly, the gel strength was about 21.00 g. In this pH range, although the pectin charge density was low, Ca²⁺ was still promoted the gel. However, the dissociation of carboxyl anion concentration could not reach the optimal amount of electronegativity carboxyl to form a stable “egg box”, gels were not so stable. In the range of pH 3.10-4.20, Calcium Bridge were formed between galacturonic acid residues and Ca^{2 +}, which made gel tend to be stable and gel strength near to 22.00 g. In the range of pH 4.20-6.62, the electrostatic repulsion was predominant between pectin chains, which generated pectin gradually depolymerization because of free dissociated -COO^- reached a maximum.【Conclusion】In the pH range of 1.78-6.30, LMP could form gels. When pH was in the range of 1.78-3.10, the pectin could be used at strongly acidic conditions and suitable for higher acidity of foods and medicines. When pH was in the range of 3.10-4.20, the stable gels could meet the food requirements of higher food gel texture. The results of this study have practical significance for the application of low methyl ester pectin in food with different pH ranges.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Effects of Hindgut Enzyme on the Enzyme Hydrolysate Gross Energy of Feedstuffs and Correlation Between Non-Starch Polysaccharides and in vitro Energy Digestibility

GAO Li-xiang, CHEN Liang, HUANG Qing-hua, ZHONG Ru-qing, ZHANG Li-lan, ZHANG Hong-fu

Scientia Agricultura Sinica. 2016, 49(13):  2612-2621.  doi:10.3864/j.issn.0578-1752.2016.13.017

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【Objective】The effects of different simulated hindgut enzymes on the in vitro dry matter digestibility (DMD) and enzyme hydrolysate gross energy (EHGE) of feedstuffs were determined using a computer-controlled simulated digestion system (SDS), and the correlation between non-starch polysaccharides (NSP) content and in vitro gross energy digestibility (GED) of feed ingredients were also analyzed to provide reference for in vitro method to simulate digestion in the stomach, small intestine and large intestine. 【Method】After the simulated digestion in the stomach and small intestine of 6 feed ingredients, 4 groups including 3 simulated hindgut enzymes (cellulase, Viscozyme and bionic enzyme) and the control with deionized water were used to stimulate the hindgut digestion with a SDS. Bionic enzymes included cellulase, xylase, β-glucanase and pectinase. Each treatment contained 5 replicates with 1 digestion tube per replicate, the DMD, GED and EHGE of corn, soybean meal, wheat bran, corn DDGS, alfalfa and soybean hull were determined. The NSP contents of these feed ingredients were measured using a gas-liquid chromatography with aiditol acetates derivatives and were also related with DMD and GED. 【Result】 The DMD (81.51 %) and EHGE (15.39 MJ·kg^-1) of corn were the highest in all ingredients, whereas the DMD (10.60 % ) and EHGE (2.42 MJ·kg^-1) of soybean hull were the lowest in the 6 feedstuffs. The DMD of corn, soybean meal and soybean hull and the EHGE of soybean meal and soybean hull were greater in the 3 simulated hindgut enzymes than in the control group (P＜0.01). The DMD and EHGE of alfalfa and DDGS were not affected by the inclusion of cellulase and the DMD and EHGE of wheat bran and DDGS were also not affected by the inclusion of Viscozyme (P＞0.05). The DMD of 6 feedstuffs and the EHGE of 6 feedstuffs with the exception of DDGS were increased with the bionic enzyme to simulate digestion in the hindgut (P＜0.01). The promotion degree of simulated hindgut enzymes on the DMD and EHGE were varied with feedstuffs. In the cellulase group, the increased DMD and EHGE were greatest in the wheat bran by 5.89% and 1.03 MJ·kg^-1, whereas were the lowest in the soybean meal by 1.26% and 0.36 MJ·kg^-1, respectively. The increasing DMD and EHGE in the Viscozyme group were the greatest in the soybean hull by 6.01% and 1.02 MJ·kg^-1, respectively, and the DMD and EHGE in the bionic enzyme group were the greatest in wheat bran by 6.59% and 1.37 MJ·kg^-1, respectively. The soluble NSP content was less than the insoluble NSP of 6 feedstuffs. Total NSP content of corn was the lowest (8.59%), whereas the total NSP content of soybean hull was the highest (75.72%). The main components of NSP included arabinose, xylose, mannose and glucose, but the 4 monosaccharide content varied in the feed ingredients. The NSP content was negatively related with the DMD, GED of feedstuffs (P＜0.05).The relationship between the DMD and TNSP contents of the 6 feedstuffs in the bionic enzyme group (R²=0.95, P＜0.01) was higher than in the cellulase group (R²=0.94, P＜0.01) and in the Viscozyme group (R²=0.93, P＜0.01). The relationship between the GED and TNSP contents of the 6 feedstuffs in the bionic enzyme group (R²=0.89, P＜0.01) was also higher than in the cellulase group (R²=0.86, P＜0.01) and in the Viscozyme group (R²=0.81, P＜0.01). 【Conclusion】 In conclusion, the inclusion of bionic enzyme to simulate hindgut digestion has a significant effect on the in vitro energy digestion of feedstuffs than the inclusion of cellulase and Viscozyme. The bionic enzyme can be used to simulate the hindgut digestion using the SDS.

DNA Barcoding of Indigenous Chickens in China: A Reevaluation

HUANG Xun-he, CHEN Jie-bo, HE Dan-lin, ZHANG Xi-quan, ZHONG Fu-sheng

Scientia Agricultura Sinica. 2016, 49(13):  2622-2633.  doi:10.3864/j.issn.0578-1752.2016.13.018

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【Objective】 The aim of this study is to determine the feasibility of utility of mitochondrial cytochrome C oxidase subunit I (COI) gene as DNA barcoding to identify indigenous chicken breeds with nearer appearances.【Method】 COI gene of 648 bp in length was obtained from nine indigenous chicken breeds of South China (Huaixiang, Qingyuan spotted, Huiyang bearded, Zhongshan shalan, Yangshan, Xinghua, Wuhua three-yellow, Wenchang and Guangxi yellow) and one commercial breed, Recessive White, with the method of direct sequencing of PCR products; while other COI sequences were downloaded from GenBank, including Chinese indigenous chickens and wild jungles and mallard (Anas platyrhynchos). These sequences were then used to analyze genetic diversity and genetic distance, construct median-joining network and phylogenetic tree based on haplotypes, as well as define breed specific haplotypes. 【Result】 COI gene of 695 bp in length was obtained after deletion of the primers sequences. And then 648 bp of standard barcoding was used for analysis. A total of 110 mutation sites were detected from 203 individuals of 10 breeds with 16.98% in all sites, of which 90 were singleton variable sites and the remaining 20 were parsimony informative sites. The average nucleotide diversity and haplotype diversity were 0.00394 (0.00349-0.00560) and 0.832 (0.763-0.905), respectively. Wuhua three-yellow chicken had the highest levels of genetic diversity, Zhongshan Shalan chicken had the second higher one, but Wenchang chicken had the lowest ones. A total of 84 haplotypes were defined, haplotype 1 had the highest frequency in nine indigenous chicken breeds. Haplotyes 9 and 5 were sharing both in indigenous and Recessive White chicken breeds, with the frequencies of 29 and 19, respectively. Each breed had its own haplotypes. Guangxi yellow, Wuhua three-yellow and Zhongshan shalan chickens had most 13 haplotypes, while Qingyuan spotted and Recessive White had least 8 haplotypes. The distribution of haplotypes of different breeds had a little of difference. For example, the haplotypes of Xinghua chicken was mostly distributed at 1, where Qingyuan spotted, Huiyang bearded and Recessive White was mostly distributed at 1 and 9, 1, 5 and 9, and 9 and 79, respectively. The genetic distance and net genetic distance between 10 breeds were ranged from 0.003 to 0.006 and from 0 to 0.003, respectively. The genetic distance among breeds was higher than those of within a breed; those of between Anas platyrhynchos and chickens were higher than 0.2. The 84 haplotyes of median-joining networks of were classified into three clusters with the characteristic of breed specific. For example, the cluster originated from in haplotype 9 had no Guangxi yellow and Wenchang chicken breeds. Other haplotypes were the descendent of 1. The Neighbor-joining tree showed that indigenous chickens in China and Gallus gallus gallus were clustered into one branch, separating from Gallus lafayetii, Gallus sonneratii and Gallus varius. No branch with breed specificity was found. 【Conclusion】The results presented herein indicated that COI gene can be used as a candidate molecular marker for elucidate genetic diversity of indigenous chickens. It’s less effectivity of utility of standard COI gene as DNA barcoding to identify indigenous chicken with nearer appearance, there is highly need to incorporate multiple molecular markers such as COI gene, Cytochrome b gene, AFLP (Amplified Fragment Length Polymorphism), SNP (Single Nucleotide Polymorphism) and breed specific appearances.

Prevalence Study and Phylogenetic Analysis of Bovine Viral Diarrhea Virus in Free-Roaming Beef Cattle in Western China

CHEN Rui, FAN Xue-zheng, ZHU Yuan-yuan, ZOU Xing-qi, XU Lu, ZHANG Qian-yi, WANG Qin, ZHAO Qi-zu

Scientia Agricultura Sinica. 2016, 49(13):  2635-2641.  doi:10.3864/j.issn.0578-1752.2016.13.019

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【Objective】Bovine viral diarrhea virus(BVDV) is commonly recognized as an important pathogen for the cattle and causes significant economic losses in the cattle industry worldwide. To assess the overall prevalence of BVDV in Chinese free- roaming beeves, the free-roaming beeves in the Western China were used in the present study by the methods of serological and bioinformatic analysis. Then, the genetic diversity of BVDV was analyzed. This study will provide a theoretical basis for the development of reasonable prevention and control, and for the vaccine development. 【Method】 A total of 1 332 sera were collected in Yunnan, Xinjiang, Gansu and Inner Mongolia from 2014 to 2015. These samples were analyzed by the commercial antibody (Ab) detection kits, and the Ab positive rate of BVDV in various regions were summarized up. Meanwhile, the positive or suspicious samples, whose S/P were less than 60%, were further detected by the Ag detection kit. Then, to isolate BVDV, the positive samples were inoculated into the MDBK cells and passaged by five generations. In addition, the primer designed based on the 5′-UTR of BVDV which has low frequency variability was used to analyze the genetic diversity of BVDV. Then the nucleotide sequences were analyzed by the bioinformatic softwares, including Sequencher4.2, BLAST, ClustalX and MEGA5.2. Finally, the genotypes and phylogenetic analysis of sequenced BVDV were confirmed based on the primers.【Result】Results showed that the average Ab positive rate of BVDV was 33.93% in the four detected provincial regions. Among them, that in the Inner Mongolia was the highest (71.43%), that in Xinjiang taking the second place (57.69%), that in Gansu was the third (16.54%) and that in Yunnan was the lowest (10.0%). The average Ag positive rate of BVDV was only 1.58%, that in Xinjiang was the highest. In this study, a total of 13 BVDV strains were isolated, they were named as Gansu 120, Mongolia 1369, Yilihuoqing 12953, Yillihuoqing 12960, Yilihuoqing 12981, Bozhoujinghe 13001, Bozhoujinghe 13023, Bozhoujinghe 13033, Xinjiangqitai 13041, Xinjiangqitai 13042, Xinjiangqitai 13191, Xinjiangqitai 13220, and Xinjiangqitai 13251, respectively. The polygenetic analysis indicated that these isolates could be mainly classified into four subtypes: BVDV-1q and BVDV-1f (isolated in Xinjiang), BVDV-1m (isolated in Inner Mongolia) and BVDV-1u which was a new subtype isolated in Gansu.【Conclusion】The serological analysis indicated that, although the free-range beef cattle overall Ab positive rate in the four provinces (autonomous regions) was lower than the national average, their situation was different. The high Ab positive rate of Inner Mongolia and Xinjiang indicated that BVDV is prevalent in the free-roaming beeves, and it is essential to minimize the damage by valuable controlling methods. The phylogenetic tree based on 5′-UTR sequence indicated that various genotypes of BVDV were existed in various regions. Overall, the diversity, cross-species transmission, cross-regional transmission and high mutation rate of BVDV make a great challenge for the immunity of vaccine. The significance of this study is to evaluate the prevalence of BVDV in free-range beef cattle through the method of healthy cattle random sample detection and analysis in various regions, and provide the reference for the prevention strategies.

RESEARCH NOTES

The Gene Expression Assays and Protein Interactions of MdJAZ1 in Apple

AN Xiu-hong, LI En-mao, LI Min, LI Zhuang, ZHANG Xiu-de, CHENG Cun-gang

Scientia Agricultura Sinica. 2016, 49(13):  2642-2650.  doi:10.3864/j.issn.0578-1752.2016.13.020

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【Objective】MdJAZ1 was isolated from apple which encoded a repressor protein in jasmonate signaling pathway. The expression of MdJAZ1 and the interactions of MdJAZ1 with other JAZ proteins or F-box protein were determined in order to lay a foundation for further functional analysis of this gene.【Method】The MdJAZ1 was selected among multiple genes which were obtained through blasting in the apple genome database using the TIFY and Jas domains. The molecular weight and isoelectric point of MdJAZ1 were analyzed by the DNAMan software, and the functional domain was analyzed using the SMART soft. The phylogenetic tree of the proteins, including MdJAZ1 and the JAZ family proteins from Arabidopsis, was constructed using the neighbor-joining (NJ) method of MEGA5.0 soft. In addition, the expression of MdJAZ1 was detected in different tissues of apple and with MeJA or wounding treatment using qPCR. The cis-acting regulatory elements of the promoter of MdJAZ1 were analyzed through PlantCARE database. Finally, the interactions of MdJAZ1 with other JAZ proteins and AtCOI1 were detected through the yeast two-hybrid assays. 【Result】The open reading frame (ORF) of MdJAZ1 was 1 149 bp in length and encoded 382 amino acids. The calculated molecular mass of MdJAZ1 was 40.536 kDa, and the isoelectric point was 9. Sequence analysis showed that MdJAZ1 protein contained the typical TIFY domain and C-terminal Jas domain. The phylogenetic relationship of MdJAZ1 was clustered with the Arabidopsis JAZ family members AtJAZ3 and AtJAZ4. qPCR analysis revealed that MdJAZ1 was expressed in all apple tissues, including roots, stems, leaves, flowers and fruits, however, the transcriptional level was significantly different in the detected tissues, with the highest expression in roots and the lowest expression in fruits. In addition, MdJAZ1 was induced by MeJA and wounding treatments, with the highest expression at 1 h. Promoter analysis showed that there were multiple putative cis-acting elements which are involved in abscisic acid response, ethylene response, and defense and stress responses; MYB binding sites were also found in the promoter of MdJAZ1 gene. The yeast two-hybrid assays showed that MdJAZ1 formed homo- and heterodimers and also interacted with AtCOI1 which encoded a F-box protein in Arabidopsis.【Conclusion】MdJAZ1 was induced by MeJA and wounding treatments. MdJAZ1 protein formed homo- and heterodimers and also interacted with AtCOI1 in the present of coronatine.