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16 September 2018, Volume 51 Issue 18

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CONTENTS

Scientia Agricultura Sinica. 2018, 51(18):  0-0. 

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Fine Mapping of Grain Length Associated QTL, qGL12 in Wild Rice (Oryza sativa L.) Using a Chromosome Segment Substitution Line

YingBin DING, LiZhen ZHANG, Rui XU, YanYan WANG, XiaoMing ZHENG, LiFang ZHANG, YunLian CHENG, Fan WU, QingWen YANG, WeiHua QIAO, JinHao LAN

Scientia Agricultura Sinica. 2018, 51(18):  3435-3444.  doi:10.3864/j.issn.0578-1752.2018.18.001

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【Objective】Fine mapping of a grain length QTL from wild rice using a chromosome segment substitution line and secondary population, exploring new genes affecting grain length and providing genetic materials and gene resources for rice breeding.【Method】Our laboratory had preliminary mapped a grain length related QTL, qGL12 using the chromosome segment substitution line (CSSL) population. On this basis, we choose one CSSL, CSSL141 which harbors qGL12 substitution segment and has significant difference of grain length compared with receptor parent 9311. CSSL141 was backcrossed with 9311. The secondary separation population was constructed for fine mapping of qGL12. Detection of length of glumes cells was performed by scanning electron microscope.【Result】CSSL141 has 4 introgressive segments from wild rice, its grain length, grain width and grain weight was significantly higher than 9311 under multi-conditions. Using CSSL141/9311 F₂ population, qGL12 was localized to the interval between RM5479 and RM28621 on chromosome 12. qGL12 affected grain length, grain width and grain weight, and the phenotypic variation explained of grain length was 44.61%. Seven polymorphic molecular marker primers in the location interval were designed, F₃ plants which had heterozygous genotype in the target interval were investigated. qGL12 was mapped to a 50 kb region between RM5479 and RM28586. Four polymorphic molecular marker primers were designed in this interval, individuals which harbor heterozygous genotype in this interval were selected for next generation F₄. Eventually qGL12 was narrowed to a 15.69 kb region between DYB9.1 and RM28586 on chromosome 12. There are 3 genes in the interval. Two candidate genes, Os12g39650 and Os12g39660, which encode a tubulin protein and a calcium-transporting ATPase respectively, with variations in their coding regions. The results of electron microscopes scanning of glumes cell showed that the length and width of 9311’s glumes cell was smaller than CSSL141, indicated that qGL12 regulate grain size of rice by controlling the size of glumes cells.【Conclusion】The wild rice grain length associated QTL qGL12 was fine mapped to a 15.69 kb region on chromosome12. qGL12 control grain length via regulate the size of glumes cells. Two candidate genes, Os12g39650 and Os12g39660, were found and would be used for further research.

Cloning and Functional Analysis of the GmWRKY148 in Soybean

ShaSha WANG, XiaoXia CUI, YanZhong HUANG, HuiDong XUAN, Na GUO, Han XING

Scientia Agricultura Sinica. 2018, 51(18):  3445-3454.  doi:10.3864/j.issn.0578-1752.2018.18.002

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【Objective】WRKY transcription factor is closely related to the biological and abiotic stress responses of plants. In order to study the function of GmWRKY148, GmWRKY148 was cloned and its expression was analyzed, then the gene was over-expressed in soybean hairy roots for analyzing resistance to Phytophthora root rot.【Method】Based on the sequence of AtWRKY44, the homologous gene Glyma.14G199800 was obtained from the root tissue of soybean Williams 82 by method of homologous cloning, named GmWRKY148. Phylogenetic analysis was performed. The transcriptional level of GmWRKY148 under four tissues and P. sojae infection was analyzed by qRT-PCR. For subcellular localization analysis, the recombinant plasmid pBinGFP2-GmWRKY148 was constructed, and transformed into onion epidermal cells by gene gun method. Transgenic soybean hairy roots were obtained by using A. rhizogenes-mediated method. The transgenic over-expressing hairy roots and empty-vector hairy roots were obtained by GFP fluorescence screening and qRT-PCR. To confirm whether GmWRKY148 is involved in the interaction between soybean and P. sojae, the positive soybean hairy roots were inoculated by P6497, and measured the lesions length, accumulation of P. sojae and the germination of oospores.【Result】The CDS of GmWRKY148 is 999 bp, encoding 332 amino acids, and the isoelectric point is 7.61. Phylogenetic analysis indicated that the WRKYs from Phaseolus vulgaris and Medicago truncatula were highly similar to GmWRKY148. Subcellular localization results showed that GmWRKY148 was located in the nucleus. Tissues expression analysis showed that GmWRKY148 had the highest expression in roots, followed in stems and leaves, and lowest in cotyledons. qRT-PCR analysis showed that the expression level of GmWRKY148 was up-regulated upon infection by P. sojae both in resistant variety Williams 82 (containing the Rps1k) and susceptible variety Williams, which has the highest expression at 24 h post infection. The transcript of GmWRKY148 is higher in Williams 82 than that in Williams. Transgenic over-expressing hairy roots (OE-GmWRKY148) and empty-vector hairy roots (EV) were inoculated with P. sojae P6497 mycelium blocks, the lesions length and the accumulation of P. sojae were compared after 24 h inoculation. The lesion length in OE-GmWRKY148 soybean hairy roots was significantly shorter than that in the EV, and the accumulation of P. sojae was significantly reduced. The rate of mycelial infection and zoospores germination was observed by microscopy at 24, 36 and 48 h after inoculated with P. sojae zoospores. Compared to the control EV, the rate of mycelia infection and zoospores germination of over-expressing soybean hairy roots was significantly decreased.【Conclusion】GmWRKY148 is a positive regulator in the interaction between soybean and P. sojae.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

A Study on the Relationship Between Water Consumption and Nitrogen Absorption, Utilization Under Sub-Soiling During the Fallow Period Plus Mulched-Sowing in Humid and Dry Years of Dryland Wheat

NianNian LI, Min SUN, ZhiQiang GAO, Juan ZHANG, HuiYu ZHANG, YanFei LIANG, QingShan YANG, ZhenPing YANG, Yan DENG

Scientia Agricultura Sinica. 2018, 51(18):  3455-3469.  doi:10.3864/j.issn.0578-1752.2018.18.003

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【Objective】The objective of these experiments was to clarify the relationship among soil water variety, nitrogen utilization and yield, and to explore emergency measures of tillage and mulched-sowing technique. 【Method】Field experiments were carried out from 2011 to 2016 in Wenxi, Shanxi province, with sub-soiling (SS) or no-tillage (NT) as the main plots and three sowing methods (Film-mulched soil hole sowing, FSH; Film-mulched sowing, FM; Drill sowing, DS) as the subplots, classification of annual type according to precipitation, in order to assess the effects of mulched-sowing under sub-soiling on relationship with water consumed and accumulation absorption and utilization in dry wheat.【Result】Under difference precipitation, the water consumption amount and its ratio was declined from sowing stage to jointing stage, but the water consumption amount and its ratio from jointing stage to mature was increased, thus water consumption during growth period was increased under sub-soiling and mulched-sowing; Nitrogen accumulation at every growth stage also was increased, especially ratio of nitrogen accumulation from jointing stage to anthesis; Pre-anthesis nitrogen translocation amount in various organs and contribution of translocation to grain was increased; Grain yield was improved significantly by 16%-30% under sub-soiling and 13%-28% under mulched-sowing, water use efficiency was improved, and nitrogen uptake efficiency and nitrogen productive efficiency was improved significantly. Difference annual precipitation and sub-soiling affected water consumption, nitrogen absorption, utilization, grain yield, water use efficiency, and nitrogen use efficiency of FHS and FM. In humid year under SS, water consumption during growth was increased, nitrogen accumulation from jointing stage to anthesis stage and contribution of leaf nitrogen translocation to grain was increased significantly, and grain yield, nitrogen uptake efficiency and nitrogen productive efficiency was increased significantly under FSH, compared with FM. However, in dry year and humid under no sub-soiling, water consumption during growth was no significantly between FSH and FM, pre-anthesis nitrogen translocation amount in various organs, contribution of stem+sheath nitrogen translocation to grain, and nitrogen accumulation amount after anthesis, finally grain yield were increased, and nitrogen uptake efficiency was increased significantly improved under FM compared with FSH. In addition, in humid year, pre-anthesis nitrogen translocation amount in various organs and nitrogen accumulation amount after anthesis was more significantly correlated with 0-120 cm water consumption from sowing stage to jointing stage, 120-300 cm water consumption at jointing stage to anthesis, 180-300 cm water consumption at anthesis to mature; In dry year, pre-anthesis nitrogen translocation amount in various organs and nitrogen accumulation amount after anthesis was more significantly correlated with 0-100 cm water consumption at sowing stage to jointing stage, 120-240 cm water consumption at jointing stage to anthesis, and 120-300 cm water consumption at anthesis to mature.【Conclusion】In conclusion, sub-soiling during the fallow period, mulched-sowing was not only increased water consumption, but also beneficial to nitrogen accumulation during growth period, ultimately significant improved yield, water use efficiency, and nitrogen use efficiency. Film-mulched soil hole sowing in humid year and film-mulched sowing in dry year increased yield and efficiency of dryland wheat under sub-soiling during the fallow period.

Reduction of Plant Height in Winter Wheat and Its Relationship with Grain Yield Under Late Frost Stress

YongFeng WU, Xin HU, DeChao REN, Ping SHI, SongCai YOU

Scientia Agricultura Sinica. 2018, 51(18):  3470-3485.  doi:10.3864/j.issn.0578-1752.2018.18.004

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【Objective】 The objective of this study was to explore the effects of late frost stress on plant height and its components in winter wheat, and to clarify the characteristics of plant height reduction and its relationship with internode length, ear length and grain yield, in order to provide a basis for establishing the evaluation index of late frost damage. 【Method】By using two frost simulation means based on the Cold Climate Chamber and the Field Movable Climate Chamber, six pot experiments and three plots experiments were carried out with the developmental progresses (floret primordia differentiation (FPD), pistil and stamen primordia differentiation (PSPD), anther connective tissue formation (ACTF), tetrad formation (TF) and heading phase) of young ear and the subfreezing treatment temperatures (-1 °C, -3 °C, -5 °C, -7 °C, -9 °C and -11 °C) as the gradients. Culm injury was investigated after each frosting treatment, and plant height and its components, and grain yield were measured at maturity. Variance analysis, regression function fitting and test method for sudden change were used to study the rule of plant height reduction, the contribution of each component to plant height, and the regression relationship of plant height and grain yield. 【Result】 (1) In the stages of PSPD to late ACTF, plant height generally showed a decreasing trend with the decrease of treatment temperature. When the treatment temperature was lower than -5℃ or so, a mutation began, and there were certain differences between individual plants and varieties. At the same treatment temperature, the greatest decline in plant height occurred in the late ACTF stage. (2) In the stages of PSPD, early ACTF and late ACTF, fourth internode length from the top (FIL), antepenultimate internode length (AIL), penultimate internode length (PIL) and ear length (EL) all showed shortening trends in different degrees under the frost stress. In these periods, the key two internodes contributing to plant height were FIL and AIL, AIL and PIL, and PIL and FIL, respectively, that showed a strongly significant correlation (P<0.001) with plant height. Correspondingly, plant height significantly decreased. (3) The regression curves between plant height and kernel number per ear, 1 000-kernel weight, and yield per plant were well fitted by the power function models. The reduction in yield per plant showed a trend from the rapid to the slow as plant height decreased. When yield per plant was reduced to 1.5 g or less because of the further frost stress, it became relatively stable as plant height continued to decrease, also, kernel number per ear did not change significantly any more at that time. 【Conclusion】 Under the simulated late frost stress, a significantly shortening trend occurred only in the imminent or elongated internodes and young ear, but not in the end of elongation. When the shortening internodes were consistent with the internodes that mainly contributed to the plant height, plant height significantly decreased. Using the shortening feature of the internodes, and the power function model between plant height and yield per plant, a new approach could be provided for the risk assessment of plant lodging in late growth and yield loss of winter wheat affected by late frost.

High Spectral Inversion of Winter Wheat LAI Based on New Vegetation Index

MeiYan SHU, XiaoHe GU, Lin SUN, JinShan ZHU, GuiJun YANG, YanCang WANG, LiYan ZHANG

Scientia Agricultura Sinica. 2018, 51(18):  3486-3496.  doi:10.3864/j.issn.0578-1752.2018.18.005

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【Objection】The purpose of this study was to analyze the effect of leaf water content on crop canopy spectra and to construct a new spectral index, so as to improve the accuracy of high spectral inversion of crop leaf area index (LAI). 【Method】 Under the support of winter wheat water-fertilizer cross test, the canopy spectral response characteristics of LAI of winter wheat under different recalcitrant cultivars, nitrogen application rates and irrigation amount were analyzed. Through the correlation analysis among the normalized differential red edge index (NDRE), water sensitivity index (WI) and LAI, the paper developed, a new vegetation index, the red-edge resistance water vegetation index (RRWVI) to inverse winter wheat LAI. Several commonly used vegetation indices were used as a reference to analyze the response ability of RRWVI to diagnose the LAI of many key winter wheat varieties. 2/3 of the measured samples were randomly selected to establish a high spectral response model of LAI based on various vegetation indices and 1/3 of the samples not involved in the modeling were used to evaluate the accuracy of the model. 【Result】 The results showed that with the advancement of growth period, the LAI of winter wheat first increased and then decreased, and different water and fertilizer treatments had a greater effect on it. After the flowering stage, the LAI of winter wheat declined significantly, and the LAI of strong gluten wheat (Gaoyou2018) was higher than that of medium-gluten wheat (Jimmy22) during the whole growth period. The spectral reflectance of winter wheat under different nitrogen levels increased with the increase of nitrogen application rate in the near-infrared band (720-1 350 nm), which was completely consistent with the nitrogen fertilizer gradient. The samples with twice-nitrogen treatment had the highest near-infrared reflectance, and the changed in spectral reflectance of winter wheat canopy under different growth stages were generally consistent. There was a high correlation between NDRE and WI in each key growth period, and the correlation between NDRE and LAI was significantly better than that of WI. The correlation between RRWVI and LAI was better than NDRE and WI. Although 8 commonly used vegetation indices are significantly correlated with LAI, RRWVI has the greatest correlation with LAI, and the coefficient of determination R2 of the fitting curve reached 0.86.【Conclusion】 By analyzing the hyperspectral inversion model of winter wheat LAI constructed by all kinds of indices, the newly constructed RRWVI achieved a more reliable inversion effect than frequently-used vegetation indices, such as NDRE and NDVI, indicating that the newly constructed red edge water-resistant vegetation index could effectively improve the accuracy of monitoring winter wheat LAI.

PLANT PROTECTION

Collation of Scientific Names of Six Maize Disease Pathogens

XiaoMing WANG

Scientia Agricultura Sinica. 2018, 51(18):  3497-3507.  doi:10.3864/j.issn.0578-1752.2018.18.006

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Fungal, bacterial and viral diseases are serious threats to maize production. In China, maize has been planted in wide areas with different agri-ecologic types and about 30 important diseases occurred annually. Because of shorter research history and limited transmission of knowledge in maize diseases, there are some confusions in using the old pathogen names of maize diseases in China, and it affects the communication about maize diseases between researches. In this paper, six scientific names of pathogens, which causing common smut, head smut, red leaf disease, northern corn leaf spot, eyespot and black bundle disease respectively, were collated based on taxonomic history of the pathogens and advantages on modern morphological taxonomy and molecular systematics. (1) The morphological and molecular characters of causal agent of common smut showed that the agent was not closely related to species of Ustilago. The name Mycosarcoma maydis, given by Brefeld in 1912, was resurrected as the valid name for maize common smut pathogen, and Ustilago maydis, a widely used name, was the synonym now. (2) Based on host selection, sporocarp morphology, host disease characteristics and result in multiple gene analyses within Sporisorium, Sphacelotheca and other related genera, Sporisorium reilianum, re-established by Langdon & Fullerton in 1978, was described as the correct name of pathogen causing maize head smut. The name, Sphacelotheca reiliana, was as one of synonyms. Because of host specificity between maize and sorghum, the maize pathogen also named as Sporisorium reilianum f. sp. zeae. (3) The virus Barley yellow dwarf virus (BYDV) is generally considered to be causal agent of maize red leaf disease. Recently, some virus strains infected maize were sequenced, and results showed clearly that Wheat yellow dwarf virus-GPV and Maize yellow dwarf virus-RMV all belong to Polerovirus genus, are pathogens causing the disease in China. (4) In spite of similar in morphology, but it is proved that genus Kabatiella is completely different from the genus Aureobasidium by multigene phylogenetic analyses. The correct name of pathogen causing maize northern corn leaf spot is Kabatiella zeae, and Aureobasidium zeae is its synonym. (5) Helmintosporium-like fungi have undergone several changes in genera. By detailed analysis on morphology, DNA sequence data, mode of life and novel metabolite production showed that Bipolaris zeicola, causing eyespot on maize, is a valid and conserved anamorph name. The name of Bipolaris was protected over Cochliobolus and approved by the Nomenclature Committee for Fungi. (6) The name Cephalosporium acremonium was confusingly used to different fungi long-time and it has lost the strict concept of criterion species. Acremonium, including more than 150 species, is also highly polyphyletic taxon with several teleomorph genera. Molecular biological characters showed the heterogeneity of the genus Acremonium. By combining molecular characteristics with morphology, old Acremonium species were reclassified into some genera, and the name Sarocladium strictum was given to the pathogen of maize black bundle disease with the synonyms Cephalosporium acremonium and Acremonium strictum.

Detection and Analysis of Fungi Carried by Maize Grain in Huang-Huai-Hai Summer Maize Region

LiuYan JIN, Ning GUO, Jie SHI, HaiJian ZHANG, ShuSen LIU, JiaQi ZHANG

Scientia Agricultura Sinica. 2018, 51(18):  3508-3519.  doi:10.3864/j.issn.0578-1752.2018.18.007

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【Objective】 The objective of this study is to determine the amount of fungi carried in maize grains in Huang-Huai- Hai summer maize region, and to provide reference for safe production, storage, processing and quarantine of maize. 【Method】A total of 720 maize ears were collected at the stages of milk-ripening and full-ripening in 90 cities or counties in 4 provinces (Hebei, Henan, Shandong, Anhui) of the Huang-Huai-Hai summer maize region, 4 full maize ears with no symptom on the surface were sampled in each city or county. All samples were tested for the amount and species of fungi carried, including external and internal tests of maize grains. Washing detection method was used to calculate the spores load on the grain surface, the spore load on the grain surface and isolation rate of each genus were calculated by counting the total number of colonies and dilution multiple. The PDA plate method was used for internal detection. Ten grains tested externally were sterilized and cultured on PDA plate. The fungi carrying rate of grains and the isolation frequency of each genus of fungi were calculated by counting the fungi carrying rate of each grain. In addition, the morphological and molecular identification of the higher frequency fungi were carried out. 【Result】The samples carried a large number of fungi. The spores load of the tested samples ranged from 0 to 1 886 per grain, with an average of 439 spores per grain at milk-ripening stage, the fungi-carrying rate of grains ranged from 0 to 65.0%, with an average of 23.6%. The spores load ranged from 18 to 2 658 per grain, with an average of 942 spores per grain at full-ripening stage, the fungi-carrying rate of grains ranged from 10.0% to 100.0%, with an average of 59.6%. The amount of fungi carried in full-ripening stage was higher than that in milk-ripening stage, but in some areas, the amount of fungi carried was still large in milk-ripening stage. There were differences in the amount of fungi carried in maize grains in different areas. The amount of fungi carried in maize grains in Henan Province was the largest, and Anhui Province had the least amount of fungi, Hebei Province and Shandong Province were in the middle and the difference was not significant. Fungal communities carried both inside and outside of maize grains included Fusarium spp., Penicillium spp., Aspergillus spp., Alternaria spp., Trichoderma spp., Rhizopus spp., Hel-minthosporium spp., Mucor spp. The isolation rate of Fusarium spp. in the grain external and internal detection was 59.1% and 36.1% respectively at milk-ripening stage, indicating that a large number of Fusarium spp. occurred at milk-ripening stage of maize. The isolation rate of Penicillium spp. and Aspergillus spp. in external grains was 8.9% and 0.7% respectively, and the isolation frequency in internal grains was 6.0% and 1.9% respectively, indicating that Penicillium spp. and Aspergillus spp. had begun to infect maize ear at milk-ripening stage. The isolation rate of Fusarium spp. in the grain external and internal detection was 71.9% and 58.5% respectively at full-ripening stage, the isolation rate of Penicillium spp. and Aspergillus spp. in external grains was 17.0% and 0.9% respectively, and the isolation frequency in internal grains was 9.3% and 2.6% respectively, indicating that Fusarium spp., Penicillium spp. and Aspergillus spp. were the main fungi carried by maize grains in Huang-Huai-Hai summer maize region. Morphological and molecular identification results showed that F. verticillioides was the dominant strain in Fusarium spp., the isolation frequency of F. verticillioides, F. proliferatum, F. graminearum was 29.7%, 25.9%, 1.3%, respectively. P. funiculosum and P. oxalicum were the dominant strains in Penicillium spp., and the isolation frequency was 5.0% and 3.6%, respectively. A. flavus and A. niger were the dominant strains in Aspergillus spp., and the isolation frequency was 1.4% and 1.2%, respectively. 【Conclusion】 The maize grains with no surface symptom carried a large number of fungi in both milk-ripening and full-ripening stages, and the amount of fungi carried in the full-ripening stage is higher than that in the milk-ripening stage. Fusarium spp. is the most frequently isolated fungi in Huang-Huai-Hai summer maize region and F. verticilliflora is the dominant strain. It is the dominant pathogen carried by local maize grains.

Identification of Pathogens Causing Brown Spot and the Role of MeJA in Disease Resistance in Stevia rebaudiana

XiaoXia CUI, HongMei SHU, Lu JIANG, XiaoLan HE, YuanYong GONG, WanChao NI, ShuQiao GUO

Scientia Agricultura Sinica. 2018, 51(18):  3520-3530.  doi:10.3864/j.issn.0578-1752.2018.18.008

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【Objective】The objective of this study is to identify the pathogenic fungus causing brown spot disease on Stevia rebaudiana, and analyze the role of MeJA in response to Alternaria to provide a basis for disease prevention and resistance breeding. 【Method】 The samples of diseased S. rebaudiana leaves were collected from the stevia production base of Fuan Town, Dongtai City, Jiangsu Province. After isolation and purification, the isolates were identified by using the methods of morphological characteristics analysis. Morphology parameters mainly included the colony and conidial morphology, conidial size. The isolates pathogenicity was tested on detached S. rebaudiana leaves. The internal transcribed spacer (ITS) rDNA region of the 7 pathogenic strains was amplified by using the fungal universal primers ITS1 and ITS4, and then the amplified product was recovered and sequenced. MEGA 7 was used to construct phylogenetic tree to confirm the pathogen species based on the rDNA-ITS sequence and the related Alternaria sequences in GenBank. The germination status of A. tenuissima conidial spores and the way they invaded the leaves after stained with trypan blue were analyzed by microscopy. The effect on the growth of A. tenuissima mycelium was analyzed by exogenous application of MeJA to potato dextrose agar (PDA) medium. To test the role of MeJA in the S. rebaudiana resistance to A. tenuissima, detached leaves were fed with MeJA and then used for infection. The qPCR method was performed to analyze the expression of JA pathway-related genes before or after inoculation with A. tenuissima in leaves of S. rebaudiana. 【Result】 Seven Alternaria strains were collected from the diseased S. rebaudiana leaves. All the strains grew on the PDA medium in a nearly circular and equal diameter radiation. The aerial hyphae were relatively developed, with white at the beginning and gradually changed to different degrees of grayish black at the later stage. The conidia are solitary or in a chain, mostly nearly spherical, inverted bar-shaped, or inverted pear-shaped, with a size of (20.5-45.5) × (6.5-16.0) μm. The isolated 7 strains were inoculated on the detached leaves of S. rebaudiana, and the pathogenicity was different. ST2, ST3 and ST7 strains led to the lesions spread rapidly with stronger pathogenicity. The rDNA-ITS sequence length of the ST2, ST3, and ST7 strains was 569, 570, and 570 bp, respectively. Phylogenetic tree analysis indicated that the similarity of ST2 and ST3 with strains KY814634.1 and DQ491089.1 (Alternaria alternata, Alternaria sp.) is between 99%-100%, and similarity between ST7 and strain HQ402558.1 (Alternaria tenuissima) is 99%. The results of trypan blue staining of S. rebaudiana leaves inoculated with A. tenuissima showed that conidia of ST7 could germinate from the head, lateral and caudal of the spores, and the A. tenuissima mycelia could invade into epidermal cells from the stomata or the epidermis intercellular. Exogenous application of MeJA could effectively inhibit the growth of A. tenuissima mycelia when the concentration of MeJA was higher than 200 μmol·L^-1. After inoculated with A. tenuissima, the lesion area of detached leaves that fed with 100 μmol·L^-1 MeJA was significantly smaller than that of the control, indicating that MeJA could enhance the S. rebaudiana resistance to A. tenuissima. JA pathway related genes were involved in the response of S. rebaudiana to A. tenuissima, the expression of LOX3 and JAR1 was up-regulated while the expression of JAZ1 and JAZ4 was down-regulated after S. rebaudiana inoculated with A. tenuissima.【Conclusion】The pathogenic fungus that caused S. rebaudiana leaf brown spot in the stevia production base of Fuan Town, Dongtai City, Jiangsu Province was identified as Alternaria. A. tenuissima mycelia can invade epidermal cells from the stomata or the epidermis intercellular space of the leaves. The resistance of S. rebaudiana leaves to A. tenuissima was enhanced after exogenous application of MeJA, which will become the potential candidates for the control of S. rebaudiana brown spot disease in the field.

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

Research of the Irrigation Mode Controlled by Cumulative Radiation on Tomato Growth and Water and Fertilizer Utilization in Greenhouse

XiaoRan WEI, RuiFeng CHENG, QiChang YANG, YongKang HE, Chen ZHANG

Scientia Agricultura Sinica. 2018, 51(18):  3531-3541.  doi:10.3864/j.issn.0578-1752.2018.18.009

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【Objective】 Under the irrigation mode controlled by cumulative radiation, the objective of this paper was to study on the effects of different irrigation amount on tomato growth, water and fertilizer utilization in tomato flowering and fruit-set stage, so as to provide the scientific basis for tomato efficient production in Chinese solar greenhouse. 【Method】 Under the condition of soil ridge substrate embedded cultivation to test “harvest” hybrid tomato by the nutrient solution drip irrigation, irrigation mode was divided into regular time interval irrigation (hereinafter referred to as “normal irrigation”, CK) and irrigation controlled by cumulative radiation, which was divided into low irrigation (T1), middle irrigation (T2) and high irrigation (T3). It researched on the irrigation modes and amount difference of growth, development, and water and fertilizer utilization in tomato flowering and fruit-set stage. 【Result】Compared with CK, the irrigation amount of T1, T2 and T3 reduced by 39.3%, 30.3% and 14.0%, respectively. The greater irrigation amount was, the higher substrate moisture content was, as follow: CK > T3 > T2 > T1. Irrigation mode controlled by cumulative radiation was better for tomato vegetative and reproductive growth, and significantly improved tomato biomass. Compared with treatment CK, the tomato biomass of T1, T2 and T3 had increased by 57.1%, 75.3% and 32.7%, respectively. Among them, the tomato biomass of treatment T2 was 102.9 g·plant^-1, which was also significantly higher than that of T1 and T3. Controlled by cumulative radiation, irrigation amount on sunny day was more than that on cloudy day, and irrigation amount on noon time was more than that on morning and afternoon, which was more consistent with the plant's demand for water and fertilizer. Besides, it saved the water and fertilizer and avoided the waste of water and fertilizer. Compared with CK, the drainage rate of T3 on sunny and cloudy day reduced by 62.5% and 72.6%, respectively. The suitable irrigation amount controlled by cumulative radiation also significantly improved tomato yield and irrigation water utilization efficiency. Compared with CK, the tomato yield of T2 was improved by 14.2%, reached to 61.3 t·hm^-2, and the irrigation water utilization efficiency was improved by 34.1%. Too little irrigation amount inhibited the production of plants. 【Conclusion】 Irrigation mode controlled by cumulative radiation could promote growth and effectively save water and fertilizer. Among them, the irrigation amount of T2 was 533.0 m³·hm^-2, which could be used as a referenced nutrient solution for tomato flowering and fruit-set stage in Chinese solar greenhouse.

Cd Accumulation and Safety Assessment of Soil-Crop System Induced by Long-Term Different Fertilization

Ke WANG, ChunLi XU, YuTing ZHANG, ZhiBin ZHENG, DingYong WANG, XiaoJun SHI

Scientia Agricultura Sinica. 2018, 51(18):  3542-3550.  doi:10.3864/j.issn.0578-1752.2018.18.010

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【Objective】 This study focused on cadmium (Cd) accumulation in soil and availability in crop induced by 23 years (1997-2013) fertilization to evaluate the extent of soil Cd contamination and the safety threshold, so that which could offer suggestions to the fertilization strategy and soil Cd safety standard of Southwest China. 【Method】 Eight fertilization treatments were employed, including only nitrogen (N), nitrogen and potassium (NK), nitrogen plus phosphorus and potassium (NPK), chemical fertilizer plus pig manure (NPK+M), chemical fertilizer plus rice straw (NPK+S), 1.5 times chemical fertilizer plus rice straw (1.5NPK+S), chloride-based fertilizers plus rice straw ((NK)_ClP+S), as well as no fertilizer control (CK). Soil available Cd, soil total Cd concentration and Cd uptake by crop among different years were measured to evaluate the Cd accumulation. 【Result】 The results showed that the soil total Cd increased with the extension of fertilization time, the total Cd accumulation in soil treated with CK, N and NK for a long time without phosphorus fertilizer increased slowly, the total Cd accumulation in soil treated with phosphate fertilizer, organic fertilizer and chlorinated fertilizer increased rapidly, and NPK+M, 1.5NPK+S and (NK)_ClP+S had more accumulation, increasing 1.18, 1.18 and 1.15 mg·kg^-1 after 23 years fertilization, respectively. Except for without phosphate fertilizer treatments, the total Cd accumulation of all the other soils above 0.6 mg·kg^-1 had the soil environmental quality risk control standard for soil contamination of agricultural land. The soil available Cd was significantly higher under all fertilization treatments than that under CK, thereinto, the content of soil available Cd increased significantly with long-term application of N, (NK) _ClP+S and 1.5NPK+S. The Cd concentration in rice grain was all raised with the increasing of fertilization time, while it didn’t exceed the national food safety standard of China (Cd≤0.2 mg·kg^-1). There were no obvious changes of wheat grain Cd concentration among different years, but only the Cd concentration in grain of (NK)_ClP+S treatment exceeded the pollutant limit standards of food of China (Cd≤0.1 mg·kg^-1). 【Conclusion】 Under the conditions of this experiment, long-term different fertilization, especially the application of phosphate fertilizer and swine manure organic manure, increased the soil total Cd content and increased the ecological risk; The long-term application of chlorinated fertilizers increased the soil available Cd content because of the decrease of soil pH, and reduced the Cd content in wheat grains. Therefore, preventing Cd from entering farmland by fertilization is an important link to ensure the safe production of agricultural products.

HORTICULTURE

Effects of Different Pollination Combinations on the Fruit Quality of ‘Fuji’ and ‘Starkrimson’ Apple

ManMan ZHANG, ZengHui WANG, YunFei MAO, ShanShan CHAI, XiaoHong ZHAO, YiChang FAN, Wei NI, ZhiQuan MAO, XueSen CHEN, Xiang SHEN

Scientia Agricultura Sinica. 2018, 51(18):  3551-3560.  doi:10.3864/j.issn.0578-1752.2018.18.011

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【Objective】The objective of this study is to explore the effect of different pollination cultivars on apple quality and volatile substance, and to provide a basis for the breeding of efficient self-pollination trees and improvement of apple fruit quality.【Method】This study investigated the apple cultivars including ‘Fuji’ and ‘Starkrimson’, which were pollinated before the blooming period by pollen from efficient pollination trees ‘Hongling’ ‘Hongjin’ ‘Hongwu’, ‘Gala’ pollen was served as a control. The content of flavonoids in fruits was measured at fruit development stage every 20 days. The external and internal quality of fruits at maturity stage were determined, such as fruit weight, fruit shape, fruit hardness, volatile substances and peel anthocyanin content.【Result】Except for titratable acid, the quality indexes of ‘Fuji’ and ‘Starkrimson’ apple fruits were higher than those of the control under the conditions of different pollination trees. After the ‘Fuji’ apple was pollinated by ‘Hongling’, its fruit shape index, hardness, anthocyanin and soluble sugar content increased significantly, 1.12, 1.15, 1.28 and 1.12 times as much as that of the control, respectively. After the ‘Starkrimson’ apple was pollinated by ‘Hongwu’, its single fruit weight, fruit shape index, anthocyanin, soluble solids, and soluble sugar content all increased significantly, 1.22, 1.12, 2.48, 1.10 and 1.11 times as much as that of the control, respectively. The titratable acid content was significantly reduced, only 75% of the control. During the whole growth period of fruit development, the content of flavonoids in apples of ‘Fuji’ and ‘Starkrimson’ treated by different pollination trees was higher than that of the control, and there were significant differences among different cultivars. At 160 d after flowering, the content of flavonoids in the fruit of ‘Fuji’ pollinated by ‘Hongling’ ‘Hongjin’ and ‘Hongwu’ increased by 19.63%, 28.72%, and 13.97%, respectively. At 120 d after flowering, the content of flavonoids in ‘Starkrimson’ apples increased by 14.18%, 15.26%, and 4.24%, respectively. The difference was significant. The relative contents of total ester volatiles in ‘Fuji’ and ‘Starkrimson’ apple fruits pollinated by ‘Hongling’ ‘Hongwu’ ‘Hongjin’ and control pollination were 50.20%, 52.03%, 42.68%, 45.10%, and 71.08%, 68.85%, 71.83%, 66.03%. After pollinated by ‘Hongling’, the content of total ester volatile substances in fruits of ‘Fuji’ and ‘Starkrimson’ increased significantly, the content of ethyl 2-methyl-butyrate was 1.14 and 203.91 times as much as that of the control, respectively. In ‘Fuji’ apple, the content of 2-methyl-butyl acetate in pollination fruits of ‘Hongling’ ‘Hongwu’ and ‘Hongjin’ was 1.73, 1.07 and 1.36 times as much as that of the control, respectively. The content of ethyl caproate and butyl acetate was 1.09, 1.12, 1.29 and 1.50, 0.77, 1.30 times as much as that of the control, respectively. In ‘Starkrimson’ apple, the content of 2-methyl-butyl acetate and ethyl caproate was 1.82, 1.27, 0.93 and 2.57, 1.15, 0.27 times as much as that of the control after pollinated by ‘Hongling’ ‘Hongwu’ and ‘Hongjin’, respectively, the content of butyl acetate was 7.83, 3.48 and 3.30 times as much as that of the control. In addition, the content of hydrocarbons of ‘Fuji’ and ‘Starkrimson’ apples after pollinated by ‘Hongling’ was higher than that of the control, which mainly showed the increase in farnesene. 【Conclusion】The appearance and intrinsic quality of ‘Fuji’ and ‘Starkrimson’ were improved significantly by high efficient pollination trees. The effect of different pollination combinations on the quality of ‘Fuji’ and ‘Starkrimson’ apples was significantly different. After pollinated by ‘Hongling’, the quality of ‘Fuji’ and ‘Starkrimson’apple fruits was significantly improved.

Effect of Exogenous Gibberellin on DNA Methylation Level and Expression of Related Enzyme Genes in Tree Peony Floral Buds

Tao ZHANG, FuHui SI, YuXi ZHANG, ShuPeng GAI

Scientia Agricultura Sinica. 2018, 51(18):  3561-3569.  doi:10.3864/j.issn.0578-1752.2018.18.012

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【Objective】 Application of exogenous gibberellin (GA) is a commonly method to break floral bud dormancy in the anti-season production of tree peony. However, the mechanism of how gibberellin plays a role in breaking dormancy of tree peony is an unsolved problem. The objective of this study is to clarify whether GA participates in dormancy regulation through epigenetic mode. 【Method】 In this study, 4-year-old ‘Luhehong’ was used as material, and 500 mg·L^-1 exogenous GA₃ was applied. Terminal buds were harvested and DNA was extracted at 0.5, 1, 3 and 5 d after GA₃ applied. The DNA of peony floral buds was extracted using CTAB method and DNA methylation level of floral buds was analyzed by HPLC technology. Three types of DNA methyltransferase genes, PsCMT, PsMET and PsDRM, and one DNA demethylase gene PsROS1 were obtained using RACE amplification based on transcriptome analysis. Bioinformatics method was performed to analyze gene sequences and possible domains, and MEGA 5.0 was used to construct phylogenetic tree. Using Actin as reference gene, the tissue expression characteristics and response to GA₃ of these genes were analyzed by qPCR method. 【Result】The open reading frame (ORF) of the three DNA methyltransferases (Dnmts) varies in length. The ORF length of PsCMT, PsMET and PsDRM is 1 118, 1 056 and 2 175 bp, respectively. The number of amino acids encoded is 372, 351 and 724, respectively. All three DNA methyltransferases have methyltransferase domains. The DNA demethylase gene PsROS1 has a long sequence with an ORF of 6 636 bp and encodes 2 211 amino acids, and there is a conserved DNA glycosylation domain in PsROS1. Phylogenetic analysis indicated that the PsCMT and PsDRM proteins in peony were closely related to the VvCMT2 protein of grape, PsMET and PsROS1 were grouped with most woody plants and located far away from tobacco. PsCMT, PsMET and PsDRM were expressed in all tissues (roots, stems, leaves, bracts, sepals, petals, stamens, carpels) at the early flowering stage of peony, and the expression level of PsCMT, PsMET and PsDRM was higher in the root of peony than that of other tissues, while the expression level of PsROS1 was the highest in carpels. Application of GA₃ dramatically accelerated buds sprouting and flowering. Bud sprouting could be observed 5 d after GA₃ application, and the germinating rate of bud was 97.5% with a 92.5% flowering rate after 60 d of GA₃ application. But in the control group, only a few buds germinated at 20 d, and the germinating rate was only 23.1% at 60 d. GA₃ significantly decreased the DNA methylation level of peony bud (P<0.01), from 38.9% before treatment to 28.7% at 5 d after treatment. GA₃ treatment increased the expression of PsCMT and PsROS1, decreased the expression level of PsDRM, but there was no significant difference in PsMET expression. 【Conclusion】GA₃ treatment induces PsROS1 expression and inhibits PsDRM expression, which leads to DNA hypomethylation and thus promotes dormancy release of peony, which may be an important way for gibberellin to play its role.

FOOD SCIENCE AND ENGINEERING

The Effects of Oxidation on Textural Properties and Water Holding Capacity of Heat-Induced Myofibrillar Protein Gel

YuLing YANG, Lei ZHOU, Yuan YOU, XiaoZhi TANG, SuMeng WEI

Scientia Agricultura Sinica. 2018, 51(18):  3570-3581.  doi:10.3864/j.issn.0578-1752.2018.18.013

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【Objective】This study was designed to investigate the influence of protein oxidation on the textural properties and water holding capacity of myofibrillar protein (MP) gel, and to reveal the root cause of gel properties changes with the degree of protein oxidation, in order to provide the theoretical basis for controlling the gel properties and the quality of chicken products.【Method】Live chickens were slaughtered and the chicken breast muscle was used to extract MP. Effects of protein oxidation on the textural properties of MP gel were studied in the lipoxygenase-linoleic acid-MP system using a texture analyzer. Water holding capacity (WHC) of MP gel was measured by high-speed centrifuge. I760 and I850/I830 measured by Raman spectroscopy were used to represent the hydrophobic interaction and hydrogen bonds of MP gel, and the potential value was determined by Zeta potential to reflect electrostatic repulsion. The change of disulfide bond was determined by the change of total sulfhydryl group (SH). The ultrastructures of the gel were observed by scanning electron microscopy. The amino acid composition and content were investigated by an amino acid analyzer. 【Result】 The carbonyl content and the degree of oxidation of MP increased with increasing linoleic acid concentration in the lipoxygenase-linoleic acid-MP system. When the content of linoleic acid increased from 0 to 2 mmol·L^-1, the gel hardness and WHC increased to the maximum, and then gradually decreased as the concentration of linoleic acid increased. Springiness slightly increased at low oxidation degree, and then decreased with more linoleic acid added. When the concentration of linoleic acid was 2 mmol·L^-1, the network of the MP gel was dense, porous and uniform in pore size. The gel pore size became larger and uneven at higher linoleic acid concentration. I760 reached the maximum at 2 mmol·L^-1 with the increase of the degree of oxidation, which indicated that hydrophobic interaction force reached maximum. The intramolecular hydrogen bonds could be formed by the three amino acid residues Ser, Glu and Cys, and the content of these three amino acids decreased with the increase of the degree of oxidation. Meanwhile, the I850/I830 of the Raman spectrum increased with the increase of the degree of oxidation and finally >1.25, indicating that the hydrogen bonds between MP molecules decreased with the increase of the degree of oxidation. After dissociation, the content of negatively charged Glu decreased with the increase of the degree of oxidation, which led to the decrease of the absolute value of Zeta potential with the increase of the degree of oxidation, indicating that the electrostatic interaction decreased with the increase of the degree of oxidation. The sulfhydryl group of Cys could form disulfide bond in the gel formation process and the content of Cys decreased with the increase of the degree of oxidation, resulting in the change of the total sulfhydryl content in the same direction, which indicated the formation of disulfide bonds in the oxidation process. The total amount of hydrophobic amino acids (Ala, Met, Val, Leu, Ile and Phe) changed with increasing degree of oxidation and reached maximum at 2 mmol·L^-1 linoleic acid, which provided evidence that hydrophobic forces reached their maximum at 2 mmol·L^-1. The principal component analysis suggested that hydrophobic interaction was the key force controlling the gel properties in the lipoxygenase- linoleic acid-MP system. 【Conclusion】 Moderate oxidation of MP helped to improve the properties of MP gels, and the gel hardness of MP reached the maximum at 2 mmol·L^-1 in the lipoxygenase-linoleic acid-MP system. The reason was that oxidation changed the composition and hydrophobic forces of MP. When the linoleic acid was 2 mmol·L^-1, the total amount of hydrophobic amino acid in MP molecule was the highest and the hydrophobic force was the largest, and the microstructure of the gel was uniform and dense, so that the texture and WHC of MP gel were the highest.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Effects of FSH Treatment on Steroidogenic Enzymes Expression and Histone H3 Modification in Pig Granulosa Cells

JinBi ZHANG, Wang YAO, ZengXiang PAN, HongLin LIU

Scientia Agricultura Sinica. 2018, 51(18):  3582-3590.  doi:10.3864/j.issn.0578-1752.2018.18.014

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【Objective】The objective of this study was to explore whether FSH treatment affect expressions of genes including steroidogenic enzymes, pituitary hormone receptors and apoptosis related genes in porcine granulosa cells, and to detect the histone H3 modification on specific gene regulation regions involved in this process. 【Method】 Firstly, ovary granulosa cells were collected using syringe extraction method from porcine ovaries and cultured in media with serum until the cells attached. After 16 h of non-serum culture, granulosa cells were treated by 5 IU?mL^-1 FSH for another 24 h culture and harvested for following experiment. Secondly, transcriptional expression changes of steroidogenic enzymes (STAR, CYP11A1, HSD3B and CYP19A1), pituitary hormone receptors (FSHR and LHR) and apoptosis related genes (XIAP and FasL) were detected using qRT-PCR method. Finally, histone H3 modification (H3K4me2, H3K4me3, H3K9ac and H3K14ac) status on regulatory regions of STAR, CYP19A1 and HSD3B genes were detected by ChIP-qPCR.【Result】 Treatment of 5 IU?mL^-1 FSH induced a significant upregulation of STAR, CYP19A1 and HSD3B genes with fold changes of 2 (P<0.01), 2.8 (P<0.0), and 3.6 (P<0.05), respectively, but had no significant effect on CYP11A1, pituitary hormone receptors FSHR, LHR and apoptosis related genes XIAP, FasL. Among the three steroidogenic genes, the histone modifications of HSD3B regulatory region were the most significant. The fold change of H3K4me2, H3K4me3, H3K9ac and H3K14ac was 14.7 (P<0.01), 13.6 (P<0.01), 19.7 (P<0.01) and 2.5 (P<0.05), respectively. H3K9ac on STAR gene regulation region decreased 11.1 (P<0.01) times. H3K4me3 on CYP19A regulation region increased 0.5 (P<0.01) times while H3K9ac decreased 10.4 (P<0.01) times. Other histone modification changes were not significant. 【Conclusion】24 h of FSH treatment enhanced the transcription levels of steroidogenic enzymes in pig granulosa cells. The up-regulation process involved H3 histone modifications in a gene-specific manner. Independent FSH treatment was not capable to induce significant effect on candidate pituitary hormone receptor and apoptosis related genes.

Mapping and Annotating of Bactrian Camel MHC Gene by Using the Comparative Genomic Approach

LiKang ZHI, Erdemtu, XiWen AN, Chao WANG, Rui WANG, Huar BAO, XiuZhen WANG

Scientia Agricultura Sinica. 2018, 51(18):  3591-3599.  doi:10.3864/j.issn.0578-1752.2018.18.015

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【Objective】The objective of this study was to locate and annotate the major histocompatibility complex (MHC) gene sequence of Bactrian camel in order to provide scientific basis for further study on Bactrian camel MHC gene. 【Method】This study used comparative genomics method. The human MHC (HLA) gene coding sequence and bovine MHC (BoLA) gene coding sequence were extracted, compared with Bactrian camel transcripts on the gene sequences through blastn, to identify the scaffolds with higher similarity. By analyzing the sequence of HLA and BoLA gene sequences on their positions on these scaffolds, multiple pieces of scaffolds were spliced to obtain the Pseudo chromosome of Bactrian camel MHC. Then, the human MHC (HLA) gene coding sequence and bovine MHC (BoLA) gene coding sequence were extracted and analyzed with the spliced scaffolds of Bactrian camels through the genomic collinearity analysis. The selected scaffolds could be judged whether or not it was accurate, based on the linear relationship between Pseudo chromosome established by lastz and HLA and BoLA genome sequences; then by analyzing the linear relationship between MHC genes in the two species, MHC gene sequences were extracted from Bactrian camel genomes, and these sequences were genetically annotated; finally, according to the obtained Bactrian camel MHC gene, the phylogenetic tree was drawn to study the evolutionary relationship between their genes. 【Result】By comparing the HLA and BoLA gene coding sequences with the Bactrian camel transcripts through blastn, three scaffolds with high similarity were identified, namely NW_011511766.1 (full-length 4.1M), NW_011515227.1 (full-length 1.2 M) and NW_011514613.1 (15K in total length), and spliced to obtain Bactrian camel MHC Pseudo chromosome; By using the lastz colinear analysis, the HLA gene sequence and the BoLA gene sequence were identified and compared with MHC gene of the Bactrian camel to obtain the colinear region. It was consistent with the spliced Pseudo chromosome, which proved that the selected scaffolds was accurate. It was found that Class-I and Class-III genes were distributed on NW_011515227.1, while Class-II genes were distributed on NW_011511766.1 and NW_011514613.1. Further analysis revealed that Class-II genes were mainly distributed in NW_011511766.1 3.5 to 4.1M position; the sequences that existed in the collinear region were extracted and subjected to blat analysis, namely aligned with the coding sequence of the MHC gene on the Bactrian camel. Results reveal that a total of 24 genes highly similar to bovine BoLA gene were identified in Bactrian camel genome, including 1 of class I gene, 10 of class II gene and 13 of class III gene. The 24 MHC genes of Bactrian camels were annotated and phylogenetic trees were mapped. The results showed that the annotated Class-I and Class-II genes were on the same branch. 【Conclusion】The method of locating and annotating the MHC gene sequence in Bactrian camel was established by comparative genomics. The MHC gene sequence of Bactrian camel was mapped to three scaffolds, 24 MHC genes were found and annotated, and the Pseudo chromosome of the MHC gene of the Bactrian camel was drawn, which laid the foundation for further study of Bactrian camel MHC gene.

Differential Expression Analysis of Long Non-Coding RNAs During the Developmental Process of Apis mellifera ligustica Worker’s Midgut

Rui GUO, SiHai GENG, CuiLing XIONG, YanZhen ZHENG, ZhongMin FU, HaiPeng WANG, Yu DU, XinYu TONG, HongXia ZHAO, DaFu CHEN

Scientia Agricultura Sinica. 2018, 51(18):  3600-3613.  doi:10.3864/j.issn.0578-1752.2018.18.016

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【Objective】Long non-coding RNA (lncRNA) plays an important role in regulation of gene expression, epigenetics and cell cycle in eukaryotes. The objective of this study is to investigate the expression profile and role of lncRNAs in the developmental process of Apis mellifera ligustica worker’s midgut. 【Method】In this study, 7- and 10-day-old worker’s midguts of A. m. ligustica (Am7, Am10) were sequenced using RNA-seq technology and strand-specific library construction method. Using Perl script, raw reads were filtered to obtain clean reads with high-quality. Bowtie tool was used to compare clean reads to the ribosome database, and TopHat2 software was employed to compare unmapped clean reads to the reference genome. CPC and CNCI softwares were utilized to predict coding capacity of the transcripts. RT-PCR was performed to identify partial lncRNAs. Investigation of differentially expressed lncRNAs (DElncRNAs) was carried out with edgeR, followed by prediction of upstream and downstream genes, for which GO and KEGG pathway enrichment analyses were performed. RNAhybrid, Miranda and TargetScan softwares were utilized together to predict target miRNAs of DElncRNAs and target genes of miRNAs, and DElncRNAs-miRNAs-mRNAs regulation networks were visualized via Cytoscape. Finally, RT-qPCR was conducted to verify reliability of the sequencing data.【Result】134 802 058 and 147 051 470 raw reads were gained from deep sequencing of Am7 and Am10, respectively, and after stringent filtration, 134 166 157 and 146 293 288 were obtained. In total, 6 353 lncRNAs were predicted, and 3 890 DElncRNAs were obtained based on expression calculation, including 2 005 up-regulated lncRNAs and 1 885 down-regulated lncRNAs. The result of RT-PCR suggested the expected signal bands could be amplified from 8 lncRNAs, implying their true existence. There were 1 793 upstream and downstream genes of DElncRNAs, which were involved in 42 GO terms, including metabolic processes, developmental processes, cellular processes, stress responses, immune system processes and so forth. They were also associated with 251 KEGG pathways, including material metabolism pathways such as carbon metabolism, purine metabolism and fatty acid biosynthesis; energy metabolism pathways such as sulfur metabolism, methane metabolism and oxidative phosphorylation; signaling pathways such as Hippo, Wnt and Notch signaling pathways; cellular immune pathways such as lysosome, endocytosis and ubiquitin mediated proteolysis; humoral immune pathways such as MAPK, Jak-STAT and NF-kappa B pathways, these results demonstrated the DElncRNAs were involved in the material and energy metabolism, cell life activity and immunity regulation in the developmental process of A. m. ligustica worker’s midgut. Further analysis showed TCONS_00020918 might play a regulatory part in the nutrient absorption and caste differentiation in the worker’s midgut. Analysis of regulation networks demonstrated that complex networks existed between DElncRNAs and target miRNAs and mRNAs, partial DElncRNAs lie in the central of the networks and link many miRNAs, and partial miRNAs could be bound by many DElncRNAs, which indicated that these DElncRNAs might play an important role during the developmental process of the worker’s midgut. Finally, 5 DElncRNAs were randomly selected for RT-qPCR assay, and the result proved the reliability of sequencing data in this study.【Conclusion】DElncRNA is widely involved in the metabolism, cellular activity and immune regulation of A. m. ligustica worker’s midgut, and plays a role as a competitive endogenous RNA (ceRNA). The results provide the necessary data support for the screening and functional study of key lncRNA.

RESEARCH NOTES

Effects of Soil Fertility on Rice Yield and Nitrogen Use Efficiency in a Red Paddy Soil

WeiFu PENG, WeiSheng LÜ, Shan HUANG, YongJun ZENG, XiaoHua PAN, QingHua SHI

Scientia Agricultura Sinica. 2018, 51(18):  3614-3624.  doi:10.3864/j.issn.0578-1752.2018.18.017

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【Objective】 Soil fertility is the basis for high rice yield in the red paddy soil. It is critical to clarify the effects of different fertility levels on crop yield and nitrogen (N) use efficiency, thereby providing scientific evidence for soil fertility improvement and appropriate fertilizer management in red paddy soil. 【Method】 A pot experiment was carried out on red paddy soils with similar texture and different fertility levels (The level of soil organic matter represented the level of soil fertility) with the ¹⁵N isotopic tracer technique. The effects of soil fertility (F_L, F_M, and F_H represent low, medium, and high fertility soils with a soil organic matter 19.9, 29.6, 38.9 g·kg^-1, respectively) and N fertilizer rate (N₀, N₁₅₀, and N₂₂₅ represent the N application rate at 0, 150, and 225 kg·hm^-2, respectively), with 9 treatments (F_LN₀, F_LN₁₅₀, F_LN₂₂₅, F_MN₀, F_MN₁₅₀, F_MN₂₂₅, F_HN₀, F_HN₁₅₀, and F_HN₂₂₅) on rice yield and its composition, N uptake and its fate were investigated. 【Result】 Increasing soil fertility and N fertilization significantly increased the number of effective panicles, yield, and total N uptake of rice. Compared with N₀, F_L, F_M, and F_H increased rice yield by 63%, 40% and 17% under N₁₅₀ treatment, respectively, with 89%, 55% and 23% under N₂₂₅ treatment, respectively. In medium and low fertility soils, increasing the N rate significantly increased rice yield, whereas no significant difference was found between the F_HN₁₅₀ and F_HN₂₂₅ treatments. Using the ¹⁵N tracing technique, we found that the uptake of fertilizer N and soil N by rice plants increased with the increasing soil fertility under the same N application rate. However, the proportion of N derived from soil (Ndfs) increased with the increasing soil fertility, while the proportion of N derived from fertilizer (Ndff) showed the opposite trend. Increasing N fertilization rate led to an increase in Ndff, while reducing Ndfs. The average N recovery efficiency (NRE) of F_L, F_M, and F_H was 42%, 48% and 52%, respectively; with the average N fertilizer residue rate of 20%, 23% and 28%, and the average N fertilizer loss rate of 38%, 29% and 20%, respectively. The NRE of F_LN₂₂₅ was significantly higher than F_LN₁₅₀ with no significant difference with F_M soils, while the NRE of F_HN₂₂₅ was significantly lower than F_HN₁₅₀. Increasing soil fertility significantly increased the contents of soil microbial biomass N, ammonium N, and fixed ammonium. 【Conclusion】 These results indicated that the higher of the soil fertility, the higher of the rice yield, the NRE and the N residual rate in the red paddy soil were, and the lower of the N loss rate was. Therefore, increasing N application rate was effective to increase both rice yield and NRE in low fertility soils. In contrast, an appropriate reduction in N application rate might benefit rice NRE and reduce fertilizer N losses without compromising rice yield.