Scientia Agricultura Sinica

Expression Differences of Dirigent-Like Protein Genes in Upland Cotton Responsed to Infection by Verticillium dahliae

GUO Bao-sheng, SHI Gong-yao, WANG Kai-hui, LIU Su-en, ZHAO Cun-peng, WANG Zhao-xiao, GENG Jun-yi, HUA Jin-ping

Scientia Agricultura Sinica. 2014, 47(22): 4349-4359. doi:10.3864/j.issn.0578-1752.2014.22.001

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【Objective】The objective of this study is to explore the Dirigent-like protein gene family and to figure out polymorphism of genes and expression differences between resistant and susceptible varieties of Upland cotton (Gossypium hirsutum) in responding to infection by Verticillium dahliae.【Method】Dirigent-like protein genes were acquired using Illumina Hiseq 2000 RNA-Seq transcriptional analysis. Multiple sequence alignment of encoding proteins was carried using online sequence analysis softwares and bioinformatics analysis tools for phylogenetic analysis. Quantitative real-time PCR with gene-specific primers was employed to identify the expression differences between resistant variety ‘Ji 79’ and susceptible variety ‘TM-1’, at 0, 1, 8, 24, 48, 72 h, and 96 h after inoculation. The quantitative reactions were performed on the ABI 7500 Real-Time PCR System with 7500 System Software (Applied Biosystems, USA) using the SYBR Green Master Mix. The comparative 2-ΔΔCt method of quantification was used with cotton GhACTIN as the reference.【Result】Totally 12 members of this gene family were found in Gossypium hirsutum, and phylogenetic analysis demonstrated 3 distinct subfamilies and 4 relatively conservative domains existed. The members of Dirigent-like protein genes were located on chromosomes 2, 3, 6, 7, 9, 10 and 13 in Gossypium raimondii, and some located in clusters on the same chromosome of D5 genome. Differential expression analysis indicated that dynamic response existed in different varieties. There were 6 members (GhDIR4, GhDIR6, GhDIR7, GhDIR9, GhDIR10 and GhDIR11) up-regulated (RQ≥2) in resistant variety Ji 79 after 1h inoculation, and RQ of GhDIR9 reached about 6.5; and 4 members (GhDIR1, GhDIR4, GhDIR7 and GhDIR11) up-regulated (RQ≥1) after 8h inoculation, only GhDIR4 up-regulated (RQ≥2) after 24h inoculation, GhDIR1, GhDIR4, GhDIR7 and GhDIR11 up-regulated (RQ≥2) after 48h inoculation; only GhDIR9 up-regulated (RQ=6.4) after 72h inoculation, up to 96 h inoculation, only GhDIR4 up-regulated (RQ≥2). GhDIR7 showed up-regulated (RQ≥2) in susceptible variety TM-1 after 1h inoculation. GhDIR7 and GhDIR10 up-regulated, and RQ of GhDIR7 increased to 325 after 8h inoculation. GhDIR4, GhDIR5, GhDIR6, GhDIR7, GhDIR9, GhDIR10 up-regulated significantly after 24h or 48h inoculation，and the RQ of GhDIR7, GhDIR9, GhDIR10 were more than 10. GhDIR4 and GhDIR7 still increased after 72h inoculation. Up to 96 h inoculation, GhDIR5, GhDIR6, GhDIR7, GhDIR9, GhDIR10 and GhDIR11 were highly expressed. So the results showed that the expression of GhDIR in response to infection by V. dahlia in resistant variety Ji 79 was earlier than that in susceptible variety TM-1. The expression of GhDIR members in susceptible variety became stronger after 8h vaccination by V. dahlia and continuously went up after 96h inoculation. Significant up-regulation of GhDIR members GhDIR4, as well as GhDIR7, GhDIR9 and GhDIR11, were observed in resistant variety, GhDIR7 and 3 other members (GhDIR5, GhDIR6 and GhDIR10) expressed in susceptible variety. And nucleotide polymorphism of 6 GhDIR genes (GhDIR1, GhDIR3, GhDIR5, GhDIR8, GhDIR10 and GhDIR12) was detected between resistant variety and susceptible variety. 【Conclusion】Dirigent-like protein plays an important role in Upland cotton in response to V. dahlia.

Analysis of the Current Situation of Accredited Maize Varieties in China

YANG Yang, WANG Feng-ge, ZHAO Jiu-ran, LIU Ya-wei

Scientia Agricultura Sinica. 2014, 47(22): 4360-4370. doi:10.3864/j.issn.0578-1752.2014.22.002

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【Objective】Variety accreditation acts as the permiting for commercialization of maize varieties, the basis for the regulation and administration of seeds market, and the reference for variety selection and right protection in China. Therefore, comprehensive analysis on accredited maize varieties according to demands of businesses such as maize variety management, and in-depth exploration of the internal link and implicit message of these variety data can provide more systematic and distinct reference to seeds management departments, scientific research institutions, seeds enterprises and farmers.【Method】 The state and provincial authorized maize variety data of China in 42 years were obtained through material search, collection and data verification, and the trend of change of maize varieties on the market in different years and regions, the repeated rate of parental inbred lines of all the accredited maize varieties and the relationship between the accreditation, protection and withdraw from promotion of maize varieties were discussed.【Result】It is shown that during the period of 1972-2013, the total number of state and provincial accredited maize varieties was 6291, among which 503 were of state level, while the present state and provincial accredited variety number was 4882, among which 332 were of state level. A total of 1726 breeding organizations have records of accredited maize varieties, and scientific research institutions and seeds enterprises accounted for 48.3% and 46.6%, respectively, with an average of 7 and 3 varieties for each organization. A total of 8754 different parental inbred lines have been used in hybridization match of all accredited maize varieties, 19.4% of which were used repeatedly. The three most frequently used inbred lines were Mo17, Chang7-2 and Dan340. A record count of 1876 of all accredited maize varieties in 42 years have been withdrawn from promotion and the average employment period of such withdrawn varieties was 13.3a. Applications for new variety property have been filed for 29.2% of all accredited maize varieties in history and approved for only 15.1% of them.【Conclusion】During the recent 10 years, the number of accredited maize varieties at all levels increased steadily year by year, so was the maize variety inventory. In terms of trend, the northern area has more accredited varieties than the southern area and the number of accredited varieties in the eastern area increased faster than that in the western area. Of all breeding organizations, scientific research institutions acted as the main entity in terms of the quantity of accredited maize varieties and the number of accredited maize varieties of large-scale seeds enterprises increased remarkably. The number of newly-named parental inbred lines has increased rapidly, so the repeating rate of them has decreased year by year. The yearly number of accredited varieties withdrawn from promotion tends to be steady and fewer varieties are overlapped in both accreditation and property applications.

Study on Homozygous Progress Evaluation of Self-Pollination in Ramie (Boehmeria Nivea L.)

TAN Long-tao, YU Chun-ming, CHEN Ping, WANG Yan-zhou, CHEN Ji-kang, WEN Lan, ZHENG Jian-shu, XIONG He-ping

Scientia Agricultura Sinica. 2014, 47(22): 4371-4379. doi:10.3864/j.issn.0578-1752.2014.22.003

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【Objective】The objective of this study was to demonstrate genetic diversity, population structure and the rate of purification of inbred progenies in ramie, and to further identify the homozygous trends. 【Method】A total of 31 pairs of SSR and 48 pairs of SRAP primers were screened out based on inbred progenies of Zhong Zhu No.1. Genetic diversity was analyzed by the homozygous locus ratio and genetic diversity parameters, population structure of inbred progenies were analyzed by using Structure, NTSYS- pcV2.1 and AMOVA softwares. Marker index(MI) was calculated to assess the discriminatory power of SSR and SRAP.【Result】 SSR and SRAP markers indicated that all genetic diversity parameters decreased obviously from S₃ to S₅, 57 and 157 polymorphic bands were amplified, the homozygous locus ratio increased by 5.2% and 4.61%, the average polymorphic loci dropped by 13 and 44, the average effective number of alleles dropped by 0.7883 and 2.1629, Nei’s gene diversity dropped by 0.1143 and 0.0684, and the average Shannon’s information index dropped by 0.0465 and 0.1207. The number of blue bands was over 65% in S₅ generation groups by SSR and SRAP with Structure software. The dispersion of S₃ generation group was higher than S₄ and S₅ generation groups, S₃ generation groups mainly contain S₄ and S₅ generation groups. Population structure and PCA by SSR and SRAP showed the uniformity of inbred progenies increased gradually. Genetic variation among populations by SSR and SRAP was larger than the variation within populations with AMOVA software, and the total variation was more than 94%. The average heterozygosity obtained using SSR (H_av=0.4689) was higher than estimated using SRAP (H_av=0.4197). Mean effective multiplex ratio was maximum for the SRAP (EMR=3.2781) followed by SSR(EMR=1.8562). Consequently, the marker index, which is the mean of the product of H_av and EMR was maximum for SRAP followed by SSR.【Conclusion】SSR and SRAP are suitable for genetic diversity and populations structure of ramie germplasm, with the increase of the number of inbred generations, the uniformity of inbred progenies increased gradually, and the average heterozygosity(H) reduced gradually.

Analysis of Physiological Characteristics about ABA Alleviating Rice Booting Stage Drought Stress

GUO Gui-hua, LIU Hai-yan, LI Gang-hua, LIU Ming, LI Yan, WANG Shao-hua, LIU Zheng-hui, TANG She, DING Yan-feng

Scientia Agricultura Sinica. 2014, 47(22): 4380-4391. doi:10.3864/j.issn.0578-1752.2014.22.004

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【Objective】The objective of the study is to reveal the drought tolerance mechanisms of different rice varieties and the physiological mechanisms about ABA reliving rice drought stress.【Method】Japonica drought tolerance rice Zaoyuxiangjing and conventional japonica rice Nanjing44 were pot planted until booting stage, and 150 mg·kg^-1 of abscisic acid (ABA) was sprayed on leaf to study the influence on rice antioxidant enzyme activity (SOD, CAT), membrane lipid peroxidation (MDA) content, net photosynthetic rate (P_n), stomatal conductance (G_s), transpiration rate (T_r), chlorophyll and soluble sugar(SS)content, biomass accumulation, yield per pot and yield components. Finally, their different biological and physiological drought resistance and response to ABA treatment were compared.【Result】During drought stress, rice maintained higher SOD and CAT activities, leading to a lower MDA content to a certain extent. They synthesized higher soluble sugar content to maintain cell osmotic adjustment. The leaf net P_n decreased consistently. G_s also showed a downward trend with increasing water stress, and T_r consistented with the G_s. Chlorophyll a synthesis was inhibited, and the content decreased. The result showed that dry matter accumulation and yield per pot were decreased significantly compared with CK. Two varieties showed significant differences in response to drought stress, and Zaoyuxiangjing was more drought resistant than Nanjing44. When ABA treated, P_n and dry matter accumulation were reduced in short time. While SOD activity was further improved, leading to reduction of MDA accumulation. Meanwhile, exogenous ABA induced stomatal closure during drought stress and reduced transpiration rate, the excessive water consumption was reduced and water use efficiency was improved, thus having a significant protective effect on rice, reduced the metabolic function of drought on rice physiological damage, and accelerated functional recovery after rehydration, keeping higher content of chlorophyll a to prevent leaf premature. The effect of ABA treatment was more significant on Zaoyuxiangjing. Its P_n, G_s and T_r recovered quickly after rehydration and a significant increase in chlorophyll a content. ABA also promoted its soluble sugar synthesis and dry matter accumulation more faster under drought stress, then improved its grains of per panicle, filled grain rate, 1000-grain weight and grain yield per pot significantly. ABA effect was weaker on Nanjing44 because of its general drought resistance. Nanjing44 was susceptible to drought stress, less sensitive to ABA, slow physiological and growth characteristics recovery. Although ABA significantly increased Nanjing 44 grains per panicle, filled grain rate and 1000-grain weight, it did not significantly increase the yield per pot.【Conclusion】The short-term “dormancy” effect on rice of ABA could relieve the physiological metabolic function damage of riceunder booting stage stress effectively, promote its functional recovery after rehydration, and mitigate the effects of drought on yield. There were differences in the response to ABA between different drought resistance varieties. The stronger drought resistance variety was more sensitive to ABA and displayed better result to exogenous ABA.

Effects of Whole Field Soil-Plastic Mulching on Spring Wheat Water Consumption, Yield, and Soil Water Balance in Semiarid Region

HOU Hui-zhi, Lü Jun-feng, GUO Tian-wen, ZHANG Guo-ping, DONG Bo, ZHANG Xu-cheng

Scientia Agricultura Sinica. 2014, 47(22): 4392-4404. doi:10.3864/j.issn.0578-1752.2014.22.005

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【Objective】The main limiting factors which affect spring wheat productivity on Northwest Loess Plateau are drought, rainfall dynamics could not meet spring wheat water needs, and low temperature in spring. How to conserve the rainwater in soil efficiently, and use it at the spring wheat growth stage, is the most important method to increase spring wheat yield in this area. The aim of the study is to reveal the effect of whole field soil plastic mulching (i.e. the whole soil surface firstly mulched by plastic, and then spread around 1cm thick soil on plastic surface) on spring wheat seasonal water consumption, yield, water use efficiency and the soil water recharge in fallow period, further to assess its effect on inter annual soil water balance in semiarid region on Northwest Loess Plateau. 【Method】The spring wheat (Triticum aestivum Lunchun 27) selected as test material, a field experiment was conducted from 2011 to 2013 on the Dingxi Experimental Station of Gansu Academy of Agricultural Sciences (104°36′ E, 35°35′ N) , which located on northwest Loess plateau. The designed three treatments are whole field soil plastic mulching and bunch seeded (FMS), whole field mulching and bunch seeded (FM), and uncovered and bunch seeded (CK). The seasonal soil water content, spring wheat biomass, yield and spring wheat yield components were recorded, as well as the rainwater fallow efficiency, evapotranspiration, water use efficiency, harvest index, and reproduction allocation index were calculated.【Result】The evapotranspiration among the three treatments were not differed significantly in 2011 and 2012, but the evapotranspiration of FMS was significantly higher than CK in 2013. From seeding to jointing stage, the FMS and FM significantly increased spring wheat evapotranspiration by 27.2% and 9.6% in dry year, 52.2% and 44.6% in wet year, respectively, as compared with CK. The evapotranspiration of FMS and FM was not significantly different at each spring wheat growth stage. However, the evapotranspiration of FMS shown an increasing trend in wet year (i.e. 2012 and 2013) as compared with FM, especially, this trend was more obvious in 2013. In fallow period, the soil water from 0 to 80 cm soil profile of FMS and FM was recharged by 25.4 mm and 18.3 mm, lower than CK by 2.2 and 9.3 mm. However, in 80-200 cm soil profile, the soil water of FMS and FM recharged by 78.0 mm and 71.0 mm, higher than CK by 30.0 mm and 23.1 mm, respectively. In the three years experimental period, the 0-200 cm profile soil water storage of FMS, FM and CK was increased by 23.8, 22.5 and 12.4mm, as compared with before sowing in 2011. The rainwater fallow efficiency of FMS was 30.5%-52.6%, higher than CK by 12.8%-109.5%, than FM by 4.5%-40.9%, spike grain number and kilo grain weight of FMS were significantly higher than CK (P＜0.05). Harvest index and reproductive allocation index of FMS was 0.42-0.49 and 0.59-0.67, averagely increased by 32.54% and 35.37% than CK, respectively. The spring wheat yield and water use efficiency of FMS reached 1 750-3 180 kg·hm^-2 and 5.5-11.5 kg·hm^-2·mm^-1, increased by 40%-220% and 27%-239% than CK, respectively. Especially, the increased extent of yield and water use efficiency was higher in dry year than that in wet year. The yield and water use efficiency of FMS were increased by 26.2% and 28.2% in the dry year (2011), and by 20.9% and 14.8% in wet year (2013), as compared with FM, the significant difference were observed. It indicated that the FMS could significantly increased spring wheat yield in wet year, and adapted drought environment in dry year, resulted in sustainable higher yield, as compared with FM.【Conclusion】FMS efficiently increased soil water storage before spring wheat sowing and early spring wheat growth stage, as well as increased spring wheat water consumption from seedling to booting stages, caused the increment of grain number per spike and reproductive allocation index, expanded sink capacity by increasing spring wheat spike grain number and kilo grain weight, resulted in higher yield and water use efficiency than FM and CK treatments. Additionally, FMS recharged the soil water from 0 to 200 cm at fallow period completely, so it is beneficial to sustain the soil water balance through years.

Study on Endosperm Development and Morphological Features of Starch Granules in Waxy Wheat Shannuo 1 and Non-Waxy Wheat Xinong 1330

YU Jing, RAN Cong-fu, LI Xue-jun, SHAO Hui, LI Li-qun

Scientia Agricultura Sinica. 2014, 47(22): 4405-4416. doi:10.3864/j.issn.0578-1752.2014.22.006

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【Objective】 The aim of this research is to study the endosperm development and morphological features of starch in waxy wheat Shannuo 1 and non-waxy wheat Xinong 1330, which will provide a theoretical basis for quality improvement of wheat breeding. 【Method】 Two wheat cultivars, Shannuo 1 (waxy wheat) and Xinong 1330 (non-waxy wheat) were used as materials in this paper. The development of endosperm cells were observed by optical microscope. In order to preserve the endosperm structure, the developing grains (5, 8, 12, 15, 18, 21, 25 and 28 d after fertilization) were immersed in glutaraldehyde and osmic acid after transected by freezing in liquid nitrogen. Before embedded with Epon812, samples were washed with phosphate buffer and dehydrated individually in a graded acetone solution. The semi-thin slices were obtained by Leica ULTRACUT slicer. Images were taken by optical microscope after stained by 1% toluidine. The cross sections of Shannuo 1 and Xinong 1330 grains and starch samples which isolated from the two wheat varieties were placed on an aluminum specimen holder, and sputter-coated with a thin ?lm of gold under vacuum condition. Samples were observed with a JEOL scanning electron microscope (JSM-6360LV, JEOL, Japan). Particle size distribution of the starch samples was measured by MASTERSIZER-2000 laser particle analyzer (Malvern UK company). All measurements were performed in triplicates. Analysis of variance (ANOVA) was performed using LSD’s test to compare treatment variations at a signi?cance level of P＜0.05 with DPS software. The data figures were performed by Sigmaplot 12.0 software. 【Result】 The results indicated that the endosperm cells of waxy wheat Shannuo 1 were smaller and developed slower than those of non-waxy wheat Xinong 1330 during grain development. In the early period of the endosperm development, the starch granules of Shannuo 1 and Xinong 1330 could be stained well by toluidine. However, in the late period of the grain development, the starch granules of Shannuo 1 could also be stained well, but Xinong 1330 can not. Compared with Xinong 1330, the endosperm of Shannuo 1 contained less protein matrix and the combination between protein and starch granules was much looser. The shape of B type starch granules of Shannuo 1 was irregular polygon, while that of Xinong 1330 was spherical. There was no significant difference in the shape of A type starch granules between Shannuo 1 and Xinong 1330. The size distribution of starch granules varied between waxy and non-waxy wheat. Volume distribution of Shannuo 1 starch granules showed a typical four-peak distribution, while that of Xinong 1330 showed a bimodal distribution. Granule surface area distribution of both cultivars indicated a typical three-peak distribution. The number distribution of granules in waxy and non-waxy starch was a typical unimodal distribution．The percentage of starch granules of Shannuo 1 and Xinong 1330 was nearly identical. In contrast, the percentage of the volume and the surface area of Shannuo 1 differed significantly from those of Xinong 1330. For A (>10 μm) type starch granules, the percentage of the volume and the surface area of Shannuo 1 were both lower than those of Xinong 1330. While for B (<10 μm) type starch granules, those of Shannuo 1 were higher than those of Xinong 1330. The volume, surface area and number of SB (<1 μm) type starch granules of Shannuo 1 were lower than those of Xinong 1330 by 1.11%, 11.60% and 9.28%, respectively, whereas the LB (1-10 μm) type starch granules of Shannuo 1 were higher than those of Xinong 1330 by 8.27%, 15.88% and 9.27%, respectively. A minority LA (>53 μm) type starch granules was found in Shannuo 1, but not in Xinong 1330. 【Conclusion】 In conclusion, the development of endosperm and the morphological features of starch granules of waxy wheat Shannuo 1 were significantly different from those of non-waxy wheat Xinong 1330 during grain development. The starch granules of LB type had considerable effects on the B type starch granules size distribution of Shannuo 1 and Xinong 1330.

Comparison and Analysis of the Field Infection Characteristics of Heterodera glycines Between Soybean and Tobacco

ZHAO Hong-hai, DING Hai-yan, WANG Feng-long

Scientia Agricultura Sinica. 2014, 47(22): 4417-4425. doi:10.3864/j.issn.0578-1752.2014.22.007

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【Objective】 The objective of this study is to observe and analyze the characteristics of population dynamics and generation cycle of Heterodera glycines (SCN) on soybean and tobacco in the same growing season of 2013, and explore the host suitability of the two host crops and the possible influences of temperature/humidity conditions on occurrence of H. glycines.【Method】 H【Result】 In soybean and tobacco soil, the 2nd stage juvenile of H. glycines emerged both from April 7to September 15, and the maximum PD occurred both in late July, but before which there were two and one unconspicuous occurrence climax, respectively. The white female first appeared in both soil on June 3, the PD of which obviously peaked two times in soybean soil in middle July and middle August, respectively, and only had one climax period of lower level in tobacco soil in June. The cyst PD peaked in both soil in middle July, afterwards it constantly maintained a relatively high level in soybean soil but declined gradually to a very low level in tobacco soil. In soybean and tobacco root, the 2nd stage juvenile first appeared both on May 19, the PD of which had three obvious peaks in soybean root occurring in late May, late June and middle July, but had only two peaks in tobacco root in middle May and late June with the former one being not obvious. The 3rd-4th stage juvenile first occurred in soybean and tobacco root on May 26and May 19, respectively, the maximum PD occurred both in late June, but there were two extra and less obvious PD climaxes on soybean in late May and middle-late July. The white female first appeared in both root on May 26. In the growing season, the nematode reproduction factors (Rf=Pf/Pi) were 5.0 and 0.5 on soybean and tobacco, respectively. Entering July afterwards, a secondary infection of H. glycines occurred on soybean, but penetration almost no longer occurred on tobacco, on the root of which a large number of empty, shrunken or small brown cysts half-embededly emerged instead.【Conclusion】In the same growing season, H. glycines occurred three generations on soybean, but only two generations on tobacco. H. glycines reproduced much better on soybean, which was much higher than tobacco in the host suitability for H. glycines. It was indicated that there was a correlation between the non-root penetrating of the 2nd stage juvenile in tobacco soil and the prolonged high soil humidity in July, and the generation progress of H. glycines seemed to be inhibited by the high temperature in July and August on soybean.. glycines samples were simultaneously and periodically collected with “Zig-Zag” sampling method and at a time interval of 6-8 d from the two crop fields in the same and fixed location. The nematodes were extracted from soil with elutriation-sieving and Baermann funnel techniques, were stained in root with sodium hypochlorite-acid fuchsin staining technique, and examined and counted under microscope. The number of each H. glycines stage in 100 g of soil or 10 g of root, population density (PD), was calculated for every ten days period in the growing season.

Blu-Rays Promote Conidiation of Metarhizium anisopliae and Expression of Related RegulatingGene fluG

WANG Miao-miao, NONG Xiang-qun, LIU Shao-fang, FAN Rong-rong, CAO Guang-chun, WANG Guang-jun, ZHANG Ze-hua

Scientia Agricultura Sinica. 2014, 47(22): 4426-4435. doi:10.3864/j.issn.0578-1752.2014.22.008

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【Objective】 The objective of this study is to determine the effect of blu-rays on Metarhizium anisopliae conidiation and related regulating gene fluG expression, and to provide a theoretical basis and technical parameters for the scale up conidia production of M. anisopliae. 【Method】 The testing strain, M. anisopliae M202 being highly virulent to white grubs, was inoculated and cultured on PDAY plates. The fungal development was observed every 12 h by microscope to confirm the developmental process of the sprout, early hyphae, exuberant hyphae, initial conidiating, exuberant conidiating and stable conidiating. The strain culture developed at 24, 48, and 72 h stages in dark were exposed to blu-rays for 0.75, 1.5, 2.25, 3, 3.75, 4.5, 5.25 and 6 h (equal to 6 804, 13 608, 20 412, 27 216, 34 020, 40 824, 47 628 and 54 432 J?m^-2), respectively. Then all of them were incubated until 10th day to finish conidiation. Each of treatments was measured conidiation by punch sampling and microsurgical counting. The conidial yield was calculated and used to assess the sensitivity of the fungal development stages to blu-rays as well as the impact of blu-rays levels on conidiation. In the meanwhile, the fluG was cloned and the real-time PCR reaction system was built. The strain culture developed at 48 h stages in dark were exposed to blu-rays at 8 gradient illuminances in 6 804-54 432 J?m^-2, respectively. Then the cultures were frozen immediately in liquid nitrogen. For the each treated culture, RNA was extracted and reverse-transcribed into cDNA, and then the expression of fluG was measured by real-time PCR. So it could assess the influence of blu-rays on gene fluG for regulating conidiation. 【Result】 Development of M. anisopliae M202 strain could be divided into several stages, including germination before 24 h, hyphae fast growing during 24-72 h with pre-conidiation at around 48 h, initial conidiation at around 60 h, rising conidiation in 72-96 h, late conidiation in 96-120 h, and conidiation mature after 120 h. After 7-10 d, the abundance of conidiation tended to constant. The results of blu-rays treatments showed that all of the 8 gradient illuminances did not cause significant difference in young mycelia of 24 h age compared with the control by no blu-rays. When older mycelia of 48 h age, growing rapidly at pre-conidiation, were exposed to the blu-rays, the conidiation could increase significantly. The optimal illuminances were in 20 412- 40 824 J?m^-2, and maximum conidiation by 34 020 J?m^-2illuminance got up to 1.50 folds compared with the control. The developmental stage of 72 h age, forming conidiation structure and increment, was most sensitive to blu-rays. The low illuminance of 6 804 J?m^-2 could significantly improve conidiation, and a wide range would be valid. For fluG, the blu-rays could significantly improve expression in the mycelia of 48 h age. With the increase of illuminance, the fluG expression was positively correlated with illuminance in less than 20 412 J?m^-2 but negatively correlated when more than 20 412 J?m^-2. The maximum expression was 2.41 folds compared with the control. Grey relationship analysis indicated that the correlation of conidiation and fluG expression was r=0.74 level when the association factor was 0.5. 【Conclusion】 Blu-rays could promote M. anisopliae conidiation and fluG expression. Sensitivity of M. anisopliae developmental stages to blu-rays was significantly different. The 48-72 h aged cultures treated with blu-rays would yield maximum conidiation at 34 020 J?m^-2 illuminance. The high correlation between conidiation and fluG expression indicated that fluG involved in the regulation of conidiation in M. anisopliae development.

Rhythm of Rice Leaf Folder (Cnaphalocrocis medinalis) to the Searchlight Trap

QI Hui-hui, ZHANG Yun-hui, WANG Jian, PENG He, ZHANG Zhi, CHENG Deng-fa

Scientia Agricultura Sinica. 2014, 47(22): 4436-4444. doi:10.3864/j.issn.0578-1752.2014.22.009

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【Objective】 The objectives of this study are to clarify the rhythm of rice leaf folder (RLF) (Cnaphalocrocis medinalis) to the searchlight trap, provide reference on monitoring and forecasting of C. medinalis, confirm the superiority of searchlight trap on monitoring migrant pests, provide data supports for biological parameters setting of trajectory analysis, and to propose a reasonable and economical plan for cutting off lighting.【Method】C. medinalis was trapped at nights by searchlight traps with automatic time-switches in Xing’an County, Guangxi Zhuang Autonomous Region in 2012 and 2013. The sampling times were 19:30-21:00, 21:00-23:00, 23:00-01:00, 01:00-03:00, 03:00-05:00, 05:00-06:00 with 6 lights, and 19:30-21:00, 21:00-22:30, 22:30-00:00, 00:00-01:30, 01:30-03:00, 03:00-04:30, 04:30-06:00 with 7 lights. The last ten days of May to the middle ten days of June were immigrant periods of C. medinalis. The end of June to the middle ten days of July were emigrant periods of C. medinalis. During different migrant periods, the days of C. medinalis amount suddenly increase were analyzed. The peak time to the searchlight trap was determined with Chi-square statistical analysis between single interval catch numbers and average interval catch numbers.【Result】The annual trapped amount of C. medinalis in 2012 and 2013 was different, but both of years had similar population dynamics with the most trapped amount of C. medinalis at 3rd generation and 4th generation. Stable rhythm of C. medinalis to the searchlight trap was obtained, but that was different in different migrant periods. Landing of migrant population occurred continually as a result of different flight abilities and atmosphere factors. For immigrant C. medinalis in 2012 and 2013, the peak time to the searchlight trap appeared mostly after mid-night, mainly during 01:00-03:00 and 03:00-05:00. Whereas, for emigrant and local C. medinalis, the peak time to the searchlight trap appeared mainly during 19:30-21:00 and 21:00-22:30. The rhythm of C. medinalis to the lights based on using searchlight trap was different from that using traditional light traps because the former can trap insects at least 500 m above ground level. C. medinalis usually took off in a large scale after sunset, the populations with shortest flight duration and longest flight duration landed during 23:00-01:00 and 05:00-06:00, respectively, and they flied for 3 h and 10 h, respectively. The source areas of C. medinalis would be more precise during trajectory analysis with the consideration of peak time to the searchlight trap.【Conclusion】The rhythm of C. medinalis to the searchlight trap was different in different migrant periods. The information of rhythm to the searchlight trap was more comprehensive than that to the traditional light traps. Considering rhythm to the searchlight trap will provide more precise time parameters for trajectory analysis.

The Correlation Analysis Between Tomato Yield, Growth Characters and Water and Nitrogen Supply

LIU Shi-quan, CAO Hong-xia, YANG Hui, LIU Shi-he

Scientia Agricultura Sinica. 2014, 47(22): 4445-4452. doi:10.3864/j.issn.0578-1752.2014.22.010

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【Objective】 A field experiment was conducted to investigate the effects of deficit irrigation and nitrogen on tomato yield and reveal the growth index of group which affected tomato yield and finally obtained the optimal model of irrigation and fertilization in solar greenhouse in Northwestern China. 【Method】The experiment was carried out to explore the effects of different water and nitrogen supplies on tomato yield, and methods of canonical correlation analysis and grey correlation analysis were used to explore the correlation of tomato yield and growth character. The treatment comprised three levels of nitrogen (N1, 150 kg·hm^-2; N2, 250 kg·hm^-2; N3, 350 kg·hm^-2) and three levels of irrigation (I1, 0.5Ep; I2, 0.75Ep; I3, 1.0Ep), Ep is the cumulative evaporation of Ф20 cm standard evaporating dish. The treatments were replicated three times in randomized complete block designs. The irrigation amount of drip irrigation system was controlled by water meter, and watering frequency was 7 to 10 d. All rotten organic fertilizer, 50% of P fertilizer, 80% of K fertilizer and part of the nitrogen (50, 150, 250 kg·hm^-2) were applied before transplanting. The amounts of P and K fertilizer were same in each treatment. And the rest of P, K and N (100 kg·hm^-2) fertilizers which were divided into the same amount of two were applied during the first and third fruit enlargement periods with the irrigation water. 【Result】The tomato yield reduced after the first rise with the increasing of applied N rates nitrogen when irrigation water was the same. Tomato yield in I2 and I3 treatments were significantly higher than I1 treatment. The tomato yield presented a trend of quadratic parabola curve with the increasing of N-rate and irrigation water amount. The canonical correlation analysis showed that the increase of plant height and leaf area may cause the increasing of fruit weight and yield reduction and the increase of root length and dry matter lead improving yield and reducing fruit weight. The grey correlation analysis method showed the order of correlation between growth traits and yield was root, plant height, leaf area, dry matter and stem diameter. 【Conclusion】The W2N2 treatment was optimal with irrigation amount of 222.8 mm and nitrogen rate of 250 kg·hm^-2. Root length and dry matter could be used as important growth indexes to control yield.

Soil Infiltration Characteristics in Fallow Period of Drip Irrigation Cotton Fields in South Xinjiang

YAO Bao-lin, LI Guang-yong, LI Fa-yong

Scientia Agricultura Sinica. 2014, 47(22): 4453-4462. doi:10.3864/j.issn.0578-1752.2014.22.011

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【Objective】The ecological environment in Tarim basin of southern Xinjiang is extremely fragile, as it’s in the arid inland river basin. As the major high quality cotton production base, mulching, drip irrigation and other water-saving irrigation technologies have relieved the contradictions of the water shortages during cotton growth period, but the winter-spring irrigation quota is relatively high. The infiltration characteristics and its influencing factors of drip irrigation cotton fields during fallow period under different surface mulching and winter irrigation quotas in south Xinjiang were studied with the aiming atproviding a theoretical basis for the spring irrigation quota and irrigation efficiency.【Method】The double-ring infiltrometer was used to measure the infiltration in a constant-head experiment. Six treatments for winter irrigation with bare fields (WIB, and the winter irrigation quota was 200 mm, the same as below), winter irrigation with high stubble fields (WIS), winter irrigation with straw mulching fields (WIM), no-winter irrigation with bare fields (NWIB), no-winter irrigation with high stubble fields (NWIS) and no-winter irrigation with straw mulching fields (NWIM) were designed. The soil infiltration characteristics and simulation infiltration process were compared and the infiltration factors were analyzed.【Result】The results showed that winter irrigation reduced initial infiltration rate, stable infiltration rate and cumulative infiltration, while WIB, WIS and WIM decreased the initial infiltration rate, stable infiltration rate and cumulative infiltration compared with NWIB, NWIS and NWIM by 22.51%, 21.49% and 12.94%, 75.86%, 27.27% and 42.86%, 61.98%, 22.18% and 29.31%, respectively. NWIM and WIM reduced the initial infiltration rate compared with bare field and high stubble fields by 14.74%, 15.20% and 4.20%, 5.96%, respectively. NWIM and WIM stable infiltration rate and cumulative infiltration showed bare fields＞straw mulching fields＞high stubble fields, stable infiltration rate of NWIB and WIB were 2.96 times, 4.39 times and 1.25 times, 1.46 times larger than straw mulching fields and high stubble fields. Cumulative infiltration NWIB was 2.13 times, 2.39 times, 2.63 times, 3.01 times and 3.07 times larger than NWIM, NWIS, WIB, WIS and WIS. In consideration of determination coefficient (R²) and root mean square error (RMSE), regression analysis of model simulation indicated that general empirical model and Kositiakov model were optimal for describing the soil infiltration process while Horton model was the second, Philip infiltration model was the third. Correlation analysis showed that the soil initial water content and soil bulk density have a negative relationship, but soil temperature and soil porosity have a positive relationship with initial infiltration rate, stable infiltration rate and cumulative infiltration. Initial moisture content of the shallow affected soil infiltration parameters obviously compared with deep soil water content. Initial infiltration rate showed a significant correlation with initial soil water content, soil temperature, soil bulk density and soil porosity at the depth of 0-10 cm soil, stable infiltration rate and cumulative infiltration showed a significant correlation with initial soil water content, soil bulk density and soil porosity at the depth of 0-10 cm soil. 【Conclusion】Winter irrigation and mulching reduced soil infiltration capacity during spring fallow period in south Xinjiang, and this method is beneficial to reducing spring irrigation quotas and improving irrigation efficiency.

Effects of Agronomic Measures on Soil Organic Carbon and Microbial Carbon Content in Cotton in Arid Region

ZHANG Qian-bing, YANG Ling, ZHANG Wang-feng, LUO Hong-hai, ZHANG Ya-li, WANG Jin

Scientia Agricultura Sinica. 2014, 47(22): 4463-4474. doi:10.3864/j.issn.0578-1752.2014.22.012

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【Objective】To reveal the regulatory mechanism of agronomic measures on soil carbon stability of the agro-ecosystem in arid region, an experiment was carried out to study the effects of agricultural practices on soil organic carbon (SOC) and microbial biomass carbon (MBC) of cotton fields, which would a provide theoretical basis for agricultural resources utilization and sustainable development in the oasis arid region.【Method】In this study, the split-split plot design method was used to arrange the treatments during cotton growth period. In the main plot treatment, two irrigation methods were designed: drip irrigation and flood irrigation. The split-plot treatment consisted of two stubble management measures: stubble-incorporated and stubble-removed. The split-split-plot treatment was composed of four fertilizer treatments: organic manure (chicken manure, which was composted before utilization, OM), N P K fertilizer (NPK), combined application of NPK fertilizer and organic manure (NPK+OM), and no fertilization (CK). The soil heterotrophic respiration intensity was measured after emergence of the cotton seedling, and soil samples were collected in the annual cotton harvest period. The soil organic carbon and microbial biomass carbon of cotton fields were measured under different agronomic measures.【Result】Both soil organic carbon and microbial biomass carbon were not significant under the condition of interaction between different agronomic measures (P＞0.05). The soil organic carbon and microbial biomass carbon at 0-20 cm surface soil were the highest, with lower distribution trends as soil layer depth increased of cotton field, and reduced to lowest at 40-60 cm soil layer in the oasis arid region. The soil organic carbon under conventional flood irrigation was 2.5%-3.0% more than that under drip irrigation under mulch, and under stubble incorporation was 2.3%-6.3% more than that under stubble removal, and as compared with CK treatment, the soil organic carbon under NPK+OM fertilization increased by 14.3%-16.8%, and the soil organic carbon of (NPK + OM) and OM treatment were significantly greater than CK and NPK treatment (P＜0.05). The microbial biomass carbon under drip irrigation under mulching was 21.9%-34.3% more than that under conventional flood irrigation method, and under stubble incorporation was 12.1%-29.4% more than that under stubble removal, and as compared with CK treatment, the microbial biomass carbon under NPK+OM fertilization increased by 83.9%-151.0%. The soil microbial quotient (qMB) was larger under drip irrigation under mulching than those under conventional flood irrigation among the irrigation treatments, and the soil microbial quotient was larger with stubble incorporation relative to removal among the stubble treatments, and the soil microbial quotient at 0-40 cm soil layer was larger than that 40-60 cm soil layer among the fertilization treatments, and the soil microbial quotient was the highest under NPK+OM fertilization, and OM, CK, and NPK, respectively. The soil microbial metabolic quotient (qCO₂) was smaller under drip irrigation under mulching than under conventional flood irrigation among the irrigation treatments, and the soil microbial metabolic quotient was smaller with stubble incorporation relative to removal among the stubble treatments, and lowest under NPK+OM fertilization (followed by OM, NPK, and CK) among the fertilization treatments, and the soil microbial metabolic quotient of (NPK + OM) treatment was significantly compared with other fertilization treatments (P＜0.05). Under the conditions of interaction between different agronomic measures, the soil microbial metabolic quotient was the lowest in the NPK+OM treatment with stubble incorporation under drip irrigation under mulching.【Conclusion】In arid region, cotton production using reasonable agronomic measures, such as water saving technology of drip irrigation under mulching, combined with application of NPK fertilizer and organic manure and straw incorporation, could not only further improve soil quality, but are also be conducive to the sustainable utilization of agricultural soils.

Effects of Water and Nitrogen Supply on the Growth and Water Use Efficiency of Cucumber（Cucumis sativus L.）Under Fertigation

LI Jing, ZHANG Fu-cang, FANG Dong-ping, LI Zhi-jun, GAO Ming-xia, WANG Hai-dong, WU Dong-ke

Scientia Agricultura Sinica. 2014, 47(22): 4475-4487. doi:10.3864/j.issn.0578-1752.2014.22.013

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【Objective】 In view of the questions of unreasonable irrigation and nitrogen in the vegetable greenhouse in semiarid northwest area, effects of water and nitrogen supply on the growth, yield, yield components, IWUE and WUE of cucumber under fertigation were investigated in order to regulate water and fertilizer scientifically and provide scientific reference for actual production. 【Method】 ‘Bonai 9-1’ was chosen as the test cultivar, the greenhouse plot experiment was subjected to three irrigation water levels [W1 (60% ET₀), W2 (80% ET₀) and W3 (100% ET₀), the irrigation of the whole growth period of cucumber were 126,152 and 177 mm, respectively] in interaction with four nitrogen fertilization levels [N0 N1 (180 kg·hm^-2), N2 (360 kg·hm^-2), N3 (540 kg·hm^-2)], and there were 12 treatments in total. Growth indexes were measured during the growing season and yield and yield components were counted after the final harvest. 【Result】 In addition to stem diameter, irrigation and nitrogen application influenced cucumber plant height, leaf area index, dry weight, yield, yield components, IWUE and WUE significantly. Irrigation had a significant positive correlation with plant height, leaf area index, fruit number, average fruit weight and yield, whereas the impact of nitrogen fertilizer on cucumber growth and yield showed different trends due to the different nitrogen levels. The impact of nitrogen fertilizer on plant height and leaf area index showed an increasing trend first and then decreasing, reached the maximum at N₂ treatment. Dry weight was slightly different, which showed under W₁ level, reaching the maximum in N₂ treatment, decreased slightly in N₃ treatment afterwards, and there were no significant difference between the N₂ and N₃ treatments. The remaining irrigation levels showed varying degrees of increase with the increasing nitrogen fertilizer. Cucumber yield also increased with the increasing nitrogen rate. When the nitrogen rate increased to N₂ level, and continued to increase, the effect of yield showed different trends under different irrigation levels. In other words, there was no significant difference among yields when nitrogen rate increased to N₂ levels and continued to increase under W₁ and W₂ levels, while difference was significant between N₂ and N₃ treatments and N₃ improved yield by 8.4% than N₂ under W₃level. Irrigation had a significant negative correlation effect on IWUE, and gained IWUE maximum 41.33 kg·m^-2 at W₁ level. The impact of irrigation on WUE showed an increasing trend and then decreasing, reached the maximum 55.82 kg·m^-2at W₂ level. Nitrogen rate had a positive correlation effect on IWUE, whereas showed different trends on WUE. In W₁ and W₂ treatments, WUE showed an increasing trend and then decreasing with the increase of nitrogen rate, reached the maximum 52.34 kg·m^-2, 55.82 kg·m^-2,respectively, at N₂ levels, while in W₃ treatment, WUE was significantly increased with the increase of nitrogen rate. The maximum yield was obtained in W₃N₃ treatment, but the WUE and IWUE were significantly lower than W₂ level. Compared with W₃N_3, the treatment of W₂N₂ reduced the irrigation amount by 16.7%, nitrogen rate by 33%, yield by only 11.3%, improved IWUE by 6.5%, and WUE by 11.1%. Path analysis on yield and growth index (height, stem diameter, leaf area index, dry weight) showed that dry weight and leaf area index had the important effects on cucumber yield improvement, which can be used for the first and second indexes of high yield.【Conclusion】Reasonable irrigation and nitrogen application not only maintained good growth characteristics of cucumber, but also obtained the maximum economic benefit. In comprehensive consideration of yield, water and fertilizer conservation, W₂N₂ treatment (80% ET₀, 360 kg N·hm^-2) was better appropriate combination of water and nitrogen under the experimental conditions.

Population Structure of ‘Candidatus Liberibacter asiaticus’ in China Revealed by SSR Markers

HUANG Ai-jun, SU Hua-nan, WANG Xue-feng, TANG Ke-zhi, LI Zhong-an, ZHOU Chang-yong

Scientia Agricultura Sinica. 2014, 47(22): 4488-4494. doi:10.3864/j.issn.0578-1752.2014.22.014

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【Objective】 ‘Candidatus Liberibacter asiaticus’ is the pathogeny of Citrus Huanglongbing. The purpose of this research was to investigate the population structure of ‘Candidatus Liberibacter asiaticus’ in China by multi SSR (simple sequence repeat) loci. 【Method】 To identify the polymorphism of SSR loci in the genome of Ca. L. asiaticus, 25 reported SSR primer sets were tested using DNA samples obtained from HLB-affected plants from 8 provinces of China. The selected SSR loci were used to analyze 285 samples by PCR and PAGE electrophoresis. The software Quantity One 4.5.0 was applied to estimate the base pairs of the amplicons. The polymorphism of 285 samples in the selected loci were evaluated via the software PopGen version 1.31, and the clustering analysis was performed by software Powermarker 3.25 and structure 2.3.4. 【Result】 A total of 5 SSR loci were identified, with Nei’s gene diversity index ranging from 0.1542 to 0.9556. The locus LasA showed high diversity. Its effective number of allele (NE) and Nei’s gene diversity (H) were 22.5 and 0.9556, respectively. Analysis on population structure of Ca. L. asiaticus of China from different geographical samples revealed that the isolates from Yunnan province had high diversity, (NE=5.7, H=0.6580). The genetic distance of different geographical populations ranged from 0.0236 to 0.5786 and genetic identity ranged from 0.5607 to 0.9767. The longest genetic distance and lowest genetic identity were identified from the geographical population between Guangxi and Sichuan. The clustering analysis indicated all isolates from China were clustered into two groups. One is from Sichuan and Yunnan, the other is from Fujian, Zhejiang, Guizhou, Guangxi, Guangdong and Jiangxi. Structure analysis also showed the populations of Yunnan and Sichuan were constituted nearly by a single group and the rest of them were made up by mixed groups. 【Conclusion】There are possible two kinds of different population structures of Ca. L. asiaticus in China by using the selected SSR markers and the results of this study will provide better insight on the origin and epidemiology of HLB in China.

The Strategy of Parameter Optimization of Bayesian Methods for Genomic Selection in Livestock

ZHU Bo, WANG Yan-hui, NIU Hong, CHEN Yan, ZHANG Lu-pei, GAO Hui-jiang, GAO Xue, LI Jun-ya, SUN Shao-hua

Scientia Agricultura Sinica. 2014, 47(22): 4495-4505. doi:10.3864/j.issn.0578-1752.2014.22.015

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Variety selection in livestock breeding occupies an important position. Genomic selection, as a novel technology in livestock breeding, has raised considerable concern. It can shorten the generation interval, speed up the genetic progress, and it can select the candidate individuals as breeding stock without phenotypic data. In 2001, Meuviwisen proposed the concept of genomic selection, which was first applied in dairy cattle. Until August 2014, there were 34 member countries of Interbull organization that had applicated genomic selection in their national dairy cattle breeding group. With the popularization and continuous promotion of genomic selection, some problems of the accuracy of genomic estimated breeding value need to be solved. Various methods of genomic selection have been proposed and more efficient models are being developed. So it has great practical significance to exploit better models and algorithm to improve the accuracy of genomic estimated breeding value. So far, there were 17 Bayesian methods that have been successively proposed. This thesis briefly introduced the classical BayesA and BayesB methods for genomic selection. BayesA assumed that all loca have effect, while BayesB supposed that a small part of locus have effect, and the percentage was extremely small. Therefore, BayesA and BayesB had different models and algorithms. After Meuviwisen proposed classic Bayesian methods, other methods were like mushrooms springing up. New Bayesian methods were based on the classical Bayesian methods, which was optimized by improving the hypothetical model and algorithm. For example, BayesC method, which was based on BayesB, optimized the π value in the model. BayesCπ and BayesDπ were the improvement of BayesC, and these two approaches assumed that marker effect variance of each locus had the same value, whereas BayesC assumed that its marker effect variance of each locus was different. BayesDπ, which was based on BayesCπ, optimized the scale parameter of inverse chi-square distribution. Bayes Lasso had the same idea with BayesA. However, its marker effects were assumed to be another distribution for Laplace, so its posterior distributions of marker effects were also changed. BayesRS method assumed that the variances of marker effect were allocated in different percentage of total genetic variance. In order to find proper hypothesis model and parameters, other Bayesian methods were also based on predecessors' research through changing the prior assumption and improving parameters of the model. At present, commonly used methods for genomic selection are classic Bayesian methods and BayesCπ, which have stabile calculation results and high accuracy of genomic estimated breeding value. In the three Bayesian algorithms, the accuracy is generally arranged into BayesB > BayesCπ > BayesA, but accuracy of genomic estimated breeding value of some traits is not the case. Compared with classical Bayesian method, parameter optimization can improve the accuracy of genome estimated breeding value to some extent. In a word, on the basis of the classical Bayesian method，for the purpose of improving the accuracy of genomic estimated breeding value, the extension of bayesian methods and its parameters optimization strategy seeked for the optimal model and parameters optimization through biological genetic algorithm combined with actual population situation. They enriched and expanded the genomic selection algorithm, and can make the genomic breeding value more reference significance. As the animal breeding process is far from the foreign breeding process in China, genomic selection can cultivate new breed, enrich the genetic resources of China and accelerate the pace of livestock and poultry breeding process. Meanwhile, the algorithm study of genomic slection and its application in China was introduced. In face of the advantages of genomic selection, whole genomic selection breeding technology is imperative. Furthermore, the main problems in current researches and the key points in future studies were also proposed, in hope of providing reference for obtaining more reliable and faster algorithm of genomic selection.

Effect of Lactobacillus reuteri I5007 on Intestinal Morphology, Disaccharidase Activity and Tight Junction Protein Expression in Newborn Piglets

YANG Feng-juan, ZENG Xiang-fang, QIAO Shi-yan

Scientia Agricultura Sinica. 2014, 47(22): 4506-4515. doi:10.3864/j.issn.0578-1752.2014.22.016

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【Objective】 The purpose of this experiment was to investigate the effects of Lactobacillus reuteri I5007 on intestinal villi maturation and intestinal barrier function in newborn piglets. 【Method】 Nine litters of 1-day-old Duroc×Landrace×Yorkshire newborn piglets and eight healthy piglets with similar body weight in each litter were selected. Piglets were randomly divided into three groups with three litters per group. The three groups were control group (oral administrated with 0.1% sterile peptone solution), the first 4 days dosed group (1-4 d, daily orally administrated with 1.2×10¹⁰ CFU of L. reuteri I5007 per piglet), and the every 4-day dosed group (1, 5, 9, 13, 17, and 21 d, daily orally administrated with 1.2×10¹⁰ CFU of L. reuteri I5007 per piglet). The trial was conducted for 21 d. During the trial, each litter of piglets were fed by a sow, respectively. Four healthy piglets closest to the average body weight from each group were fasting weighted, anesthetized and then slaughtered on d 7, 14 and 21. The small intestine was carefully dissected from the mesentery and segments (the same location in all piglets), including duodenum, jejunum and ileum, were collected. Growth performance, intestinal morphology, disaccharidase activity and expression of tight junction (TJ) proteins were measured. 【Result】 Oral administration with L. reuteri I5007 had no significant effect (P＞0.05) on growth performance, but had impacts on piglet intestinal morphology at different time points. On d 7, duodenal villus height, villus height/crypt depth and goblet cells density and ileal goblet cells density of piglets in every 4-day dosed group were increased significantly (P＜0.05) compared with those of control piglets. Piglets in first 4 days dosed group had distinct higher (P＜0.05) duodenal villus height/crypt depth and goblet cells density and ileal goblet cells density than control piglets, but not villus height/crypt depth (P＞0.05). On d 14, administration with L. reuteri I5007 in every 4-day dosed group robustly increased (P＜0.05) duodenal villus height/crypt depth and ileal goblet cells density. Ileal goblet cells density of piglets in first 4 days dosed group was significant higher (P＜0.05) than that of control piglets, but lower (P＜0.05) than that of piglets in every 4-day dosed group. On d 21, piglets of every 4-day dosed group had an increased goblet cells density (P＜0.05) in both duodenum and ileum. Compared with control group, administration of L. reuteri I5007 in first 4 days dosed group also augmented (P＜0.05) ileal goblet cells density, but reduced (P＜0.05) duodenal villus height/crypt depth. In addition, piglets in first 4 days dosed group had lower (P＜0.05) duodenal villus height/crypt depth and goblet cells density than those of piglets in every 4-day dosed group. For disaccharidase activity, piglets in every 4-day dosed group had higher (P＜0.05) both sucrase and maltase activities in jejunum than those of control piglets. Only jejunal maltase activity of first 4 days dosed piglets was enhanced (P＜0.05) compared with control piglets, while jejunal sucrase and maltase activities were significantly lower (P＜0.05) than those of every 4-day dosed. Furthermore, piglets in every 4-day dosed group had significant higher expression of Claudin-1, Occludin and ZO-1 in both jejunal and ileal epithelial cells (P＜0.05). Administration of L. reuteri I5007 in first 4 days dosed group also promoted (P＜0.05) expression of jejunum epithelial Claudin-1 and ZO-1 and ileum epithelial Claudin-1. However, the level of jejunum epithelial Occludin of first 4 days dosed piglets was obviously lower (P＜0.05) than that of every 4-day dosed piglets. Besides, the levels of Claudin-1 (P = 0.1) and ZO-1 (P = 0.1) in ileum epithelium of first 4 days dosed piglets had a trend of decrease compared with every 4-day dosed piglets. 【Conclusion】 Every 4-day administration of L. reuteri I5007 in newborn piglets can promote the maturation of intestinal villi, improve jejunal disaccharidase activity, and increase expression of TJ protein of intestinal epithelial cells. Thus, L. reuteri I5007 may play a role in protecting or augmenting intestinal barrier function in newborn piglets.

Influence of Ammonia Concentration on Growth Performance and Meat Quality of Broilers

LI Cong, LU Qing-ping, TANG Xiang-fang, ZHANG Ji-ze, DING Ze-min, ZHANG Hong-fu

Scientia Agricultura Sinica. 2014, 47(22): 4516-4523. doi:10.3864/j.issn.0578-1752.2014.22.017

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【Objective】This study was conducted to investigate the influence of ammonia concentration on growth performance and meat quality of broilers.【Method】The experiment was conducted in four chambers of the State Key Laboratory of Animal Nutrition. Three hundred and twenty 21-day-old Arbor Acres broilers with similar initial body weight were divided randomly into 4 groups for different treatments, with 4 replicates in each group and 20 broilers for each replicate. Each treatment of broilers was placed in a separated, environmentally controlled chamber, and ammonia concentrations in the four chambers were metered continuously to maintain at (3±3), (25±3), (50±3) and (75±3) µL·L^-1, respectively. The broilers were net-rearing and provided ad libitum access to water and diets. The experiment lasted for three weeks. Broilers’ feed intake and health status were recorded, broilers were weighed according to replicate to get the growth performance at the age of 32 and 42 days, at the same time, three broilers were selected and slaughtered from each replicate to measure slaughter performance and meat quality.【Result】 During the age from 22 to 32 days, when ammonia concentration was at 50, 75µL·L^-1, ADG and ADFI of broilers were significantly decreased compared with the control group(P＜0.05); ammonia concentration did not significantly affect F/G of broilers(P＞0.05). During this period, ammonia concentration did not significantly affect the dressing percentage, eviscerated carcass percentage and half-eviscerated carcass percentage of broilers (P＞0.05). During the age from 33 to 42 days, compared with the control group, ADFI of broilers was significantly decreased when ammonia concentration was 25-75 µL·L^-1 (P＜0.05); ammonia concentration did not significantly affect ADG and F/G of broilers (P＞0.05). During the whole experiment period, compared with the control groups, ADG and ADFI of broilers in all three experiment group had a significantly decrease(P＜0.05), F/G of broilers was significant increased(P＜0.05); abdominal fat percentage of broilers had a trend of rising with the increase of ammonia concentration. Compared with the control group, when ammonia concentration was at 75µL·L^-1, abdominal fat percentage of broilers was significantly improved(P＜0.05); ammonia concentration did not significantly affect the breast meat pH(after 24 h),color L* value, color a* value and color b* value (P＞0.05). Compared with the control groups, drip loss rate of breast meat in all three experiment group had a significant increase (P＜0.05). 【Conclusion】These results indicate that ammonia concentration at 25µL·L^-1 in chamber can inhibit the growth performance and meat quality of broilers, and with the increase of ammonia concentration, the influence become worse, abdominal fat percentage and drip loss rate of breast meat of broilers can be significantly improved when they are under the high ammonia concentration conditions, so, by integrating the foregoing study outcomes, the author suggested that ammonia concentration in chamber should be controlled within 25µL·L^-1.

Cloning and Function Analysis of a MaDFR Gene from Mulberry

LI Jun, ZHAO Ai-chun, UMUHOZA Diane, WANG Xi-ling, LIU Chang-ying, LU Cheng, YU Mao-de

Scientia Agricultura Sinica. 2014, 47(22): 4524-4532. doi:10.3864/j.issn.0578-1752.2014.22.018

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【Objective】In this study, MaDFR was isolated from mulberry (Morus alba L.) and transformed into tobacco for function analysis of flavonoids biosynthesis and providing application basement. 【Method】Fragment of MaDFR was obtained by RT-PCR and transformed into tobacco by using Agrobacterium tumerficians-mediated transformation approach. Plants with kanamycin resistance were obtained for identification by using genome PCR and insertion sites analysis by reverse PCR. The expression levels were determined by qRT-PCR. The concentration of total flavonoids was analyzed by AlCl₃ and the antioxidant activity of acid hydrolysis and acid hydrolysis was showed by using DPPH scavenging ability. HPLC was used for identification of anthocyanins in tobacco. 【Result】The full-length CDS sequence of MaDFR obtabined from Zhongsang 5801 is 1026 bp in length encoding a putative protein about 341 amino acids with a typical NADPH binding site. A transgenic vector based on pBI121 was constructed and transformed into tobacco by Agrobacterium tumerficians-mediated transformation approach. Seven plants with kanamycin resistance were obtained. Four positive plants were identified by genome PCR and reverse PCR. In transgenic plants MaDFR showed different expression levels. There were significant increases of total flavonoids in transgenic plants, DFR-2(19.8%), DFR-4(30.4%), DFR-5(14.2%), and DFR-7 (27.6%), respectively. There was a large difference in the antioxidant activity between control and transgenic plants, the antioxidant activity of total flavonoids in DFR-2 and DFR-4 showed significant increase by 5 times, while the antioxidant activity of DFR-5 and DFR-7 showed no significant difference compared to control. The antioxidant activity of total flavonoids before acid hydrolysis showed a well correlation with the expression levels of MaDFR in tobacco. The antioxidant activity of acid hydrolysis of total flavonoids in transgenic plants showed a well correlation with the contents of total flavonoids. The overexpression of MaDFR improved the content of anthocyanins in transgenic tobacco corolla, DFR-4(15%), DFR-5(100%) and DFR-7 (100%), respectively, but could not change the components of anthocyanins.【Conclusion】The overexpression of MaDFR in tobacco increased the content and antioxidant activity of total flavonoids. MaDFR also deepened the color of tobacco corolla by improving the content of anthocyanins, and thus playing an important role in biosynthesis of anthocyanins, especially in cyanidin.

Artificial Rearing and Morphological Determination of the Diploid Honey Bee in Apis cerana cerana

GAN Hai-yan, LI Shu-yun, ZENG Zhi-jiang, YAN Wei-yu

Scientia Agricultura Sinica. 2014, 47(22): 4533-4539. doi:10.3864/j.issn.0578-1752.2014.22.019

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【Objective】 The objective of this experiment is to study artificial rearing and determination of morphological index of diploid drones, and to reveal the biological characters of Apis cerana cerana. 【Method】 Queens were firstly reared by the method of larvae double-transferring, and then anaesthetized with CO₂. The colony were fed with sucrose for stimulative feeding. Unfertilized eggs were laid by queen, and they were marked by painting after emergence. Haploid drones of sexual maturity were caught for insemination with their mother queen. The queen was then controlled to lay eggs on an empty frame. Larvae developed from the eggs were transferred to incubator (RH: 95%; T: 35℃) for artificial rearing. Larvae in first 3 days were fed with diet containing 90% royal jelly and 10% ddH₂O. During 4-6 days old, the larvae food was changed to 50% royal jelly, 6% fructose, 6% glucose, 1% yeast extract and 37% ddH₂O. From 7 days old to defecating period the diet is constituted of 43% royal jelly, 9% fructose, 9% glucose, 1% yeast extract and 38% ddH₂O. The cell ploidy of cultivated drones was determined using flow cytometry instrument and morphological index of haploid and diploid drones in colony were measured. 【Result】 The total emerged rate in honeybee of artificial feeding was low (36% in average), in which 26.9% were drones. There were 92% of emerged drones reared in vitro and 82% developed in natural colony drones were diploid examined by the cell ploidy. According to the morphological index investigation, the birth weight and reproductive organs of diploid drones (99.78 and 6.05 mg, respectively) were significantly smaller than that of haploid drones (105.64 and 7.02 mg, respectively), and in other indexs (the fore wing length, the fore wing width, the number of the hooks on the wing, etc) the difference was not significant. 【Conclusion】 After inbreeding, the queens (A. cerana cerana) can produce the diploid drones, and some larvae can reach adult stage. And some morphological differences exist between them and haploid drones.

The Polyphenols Composition of Lycium ruthenicum Murr. from Different Places

YAN Ya-mei, DAI Guo-li, RAN Lin-wu, LUO Qing, LI Xiao-ying, QIN Ken, WU Peng-ju, ZENG Xiao-xiong, CAO You-long

Scientia Agricultura Sinica. 2014, 47(22): 4540-4550. doi:10.3864/j.issn.0578-1752.2014.22.020

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【Objective】 Lycium ruthenicum 【Method】The contents of total polyphenols, total flavones and total anthocyanins were analyzed using spectrophotometer and the composition of the polyphenol extracts was analyzed using HPLC and HPLC-MS. Hierarchical cluster analysis (HCA) was also used to classify the tested results. 【Result】 Total contents of polyphenols, flavones and anthocyanins of the L. ruthenicum Murr.s were 8.25-87.77 mg GAE·g^-1 dry fruit, 18.03-60.44 mg RE·g^-1 dry fruit, and 8.21-31.46 mg·g^-1 dry fruit, respectively. The samples from Wutumeiren Gomud of Qinghai, Ganhezi of Xinjiang, Baluntai town of Xinjiang, and Helan12-1 of Ningxia had the higher value of total polyphenols, total flavones and total anthocyanins. Through HPLC-ESI-MS analysis, a total of 19 polyphenols were identified in the tested L. ruthenicum Murr, including 7 acylated anthocyanins, and petunidin-3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside was found to be the main anthocyanin in L. ruthenicum Murr. Twenty polyphenols were found in all kinds of L. ruthenicum Murr, while Petunidin-3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside was the main composition of the polyphenols in fruits of L. ruthenicum Murr. The polyphenols fingerprints of L. ruthenicum Murr. from 26 different places were similar, excluding a few of samples. The results of HCA according to the contents of polyphenols, flavones and anthocyanins in L. ruthenicum Murr. were clustered into7 kinds. In the sixth and seventh kinds, all the contents of polyphenols, flavones and anthocyanins were higher(57.74-87.77 mg·g^-1, 41.52-55.46 mg·g^-1, 12.25-28.54 mg·g^-1, respectively) in the L. ruthenicum Murr. samples from Zhongqu Mingqin of Gansu, Kashi of Xinjiang, and 12-01 Helan of Ningxia. Especially, the sample from the Ganhezi of Xinjiang would be the good elite material using the index of the contents of polyphenols and anthocyanins. The result of clustering analysis according to HPLC shared peaks’ area of L. ruthenicum Murr. was clustered into five kinds according to the 12 shared peaks’ area, among which the fourth kinds Helan 12-1, Wutumeiren Gomud of Qinghai, Ganhezi of Xinjiang samples’ relative peak area was high, and as the main component, peak 22 relative peak area was between 2 350.84 and 3 092.94 . The samples from Baluntai town of Xinjiang as a separate kinds, its main component peak 22 relative peak area reached up to 3 579.96. 【Conclusion】 The compositons and total polyphenols contents in fruits of L. ruthenicum Murr. from different places are different, but most of the samples are good resources for development of healthy food which are rich in polyphenols, exceptionally, acylation- petunidin. Murr. is grown in the northwest of China, and its fruits is used in medicinal foods for treatments of heart disease, abnormal menstruation and menopause. In the past few years, the polyphenols (especially anthocyanins) are proved to be the main effective components in Lycium ruthenicum Murr. Composition determination and evaluation of anthocyanin and other polyphenols of wild L. ruthenicum fruit in different origin, so as to provide the basis for the breeding and utilization of it.

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