Scientia Agricultura Sinica

Identification of the Differentially Expressed Proteins Between Heat-Tolerant and Heat-Sensitive Rice Responding to High-Temperature Stress at the Early Milky Stage

LIAO Jiang-Lin, SONG Yu, ZHONG Ping-An, ZHOU Hui-Wen, ZHANG Hong-Yu, HUANG Ying-Jin

Scientia Agricultura Sinica. 2014, 47(16): 3121-3131. doi:10.3864/j.issn.0578-1752.2014.16.001

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【Objective】The objective of this study is to identify the differentially expressed proteins and their biological functions involving in rice grain responding to high temperature stress at the early milky stage.【Method】The heat-tolerant rice line XN0437T and the heat-sensitive rice line XN0437S were used as plant materials. The tub-planting method and conventional cultivation way were used to culture the rice. To ensure that only uniformly developed samples were used for analysis, rice ears were labeled on the same heading date and the panicles on the labeled ears were further labeled on the same flowering date. On the 10th day after heading, plants with the same label were transferred to chambers and maintained at a temperature of 38.0/25.0 ± 0.5℃ (treatment / control) for 1 day, 3days and 5days. After the initiation of high temperature stress at the early milky stage of rice, rice grains from the same region (middle to bottom part) of labeled ears were harvested at three different time points. The total proteins from harvested sample were extracted by the trichloroacetate (TCA) - acetone precipitation method and the protein profiles of rice grain were set up by two-dimensional electrophoresis (2-DE). The proteins in rice responding to high temperature stress (PRHT) were firstly identified by comparing the 2-DE protein profiles between treatment and its parallel control for heat-tolerant and -sensitive lines, respectively. The differentially expressed proteins were then confirmed by comparing the volume of PRHT from heat-tolerant and -sensitive lines. The functions of these differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis and protein database searching. 【Result】Twenty-seven differentially expressed protein spots were found and showed more than 2-folds difference in PRHT volume between the heat-tolerant and -sensitive lines, and 25 proteins were identified by MS and database searching. The 25 identified proteins were involved in biological synthesis (10 proteins), energy metabolism (4 proteins), oxidation (7 proteins), emergency response (3 proteins) and transcriptional regulation (1 protein). 【Conclusion】The expression patterns of the rice grain proteins, which are involved in biological synthesis, energy metabolism, oxidation, emergency response and transcriptional regulation, were influnced by high temperature stress at early milky stage of rice grain. The expression patterns of these proteins were showed variable according to the difference in rice genetype (heat-tolerant and -sensitive lines) and extent (days) of high temperature treatment.

Genomic DNA Methylation Polymorphism Analysis of Cotton Under NaCl and Na2CO3 Stress

LU Xu-Ke, WANG De-Long, YIN Zu-Jun, WANG Jun-Juan, FAN Wei-Li, WANG Shuai, ZHAO Xiao-Jie, ZHANG Tian-Bao, YE Wu-Wei

Scientia Agricultura Sinica. 2014, 47(16): 3132-3142. doi:10.3864/j.issn.0578-1752.2014.16.002

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【Objective】 The objective of the study is to research the change of genomic DNA methylation under different types of salt and to compare the variation of DNA methylation between leaves and roots, which can help us make it clear about the relation between DNA methylation and the salt tolerance in cotton.【Method】In the study, salt-tolerant variety Zhong9806 and salt-sensitive variety ZhongS9612 were used as experimental materials, which were treated with NaCl and Na2CO3 at concentration of 0.4%, respectively. DNA extracted from control materials and the treatment materials were used for double digestions, ligation reactions, pre-amplification reactions and selective amplification reactions, then MSAP technology (Methylation sensitive amplified polymorphism) was used to explore the change of DNA methylation between the control and treatment materials in cotton seedlings. The differentially methylated DNA fragments were isolated from the polyacrylamide gel, sequenced and BLAST analysis in NCBI library. Real time-PCR technology was used to test the expression quantity of polymorphic fragments in cotton seedlings.【Result】The results of salt stress indicated that the effects of different salt stresses on the growth of cotton seedlings were different. Neutral salt NaCl at 0.4% concentration had a relatively small effect on cotton seedling，and the morphological characters of different tissues changed a little, but 0.4% alkaline salt Na2CO3 had a bigger harm, resulted in the cotyledon soft, and the basal part of stem and roots of cotton seedlings black. MSAP analysis showed that after NaCl treatment, the genomic DNA methylation level of leaves of Zhong9806 and ZhongS9612 were 23.5% and 27.7%, in which full methylated ratio was 20.3% and 22.9%, respectively. The methylation level of roots of Zhong9806 and ZhongS9612 was 24.7% and 27.1%, in which full methylated ratio was 19.6% and 21.6%, respectively. After Na2CO3 treatment, the methylation level of leaves of Zhong9806 and ZhongS9612 were 28.9% and 28.1%, in which full methylated ratio was 24.3% and 24.5%, respectively. The methylation level of roots of Zhong9806 and ZhongS9612 was 25.7% and 27.6%, in which full methylated ratio was 21.5% and 24.0%, respectively. Genomic DNA methylation levels were different between leaves and roots. With the salt type changed from neutral salt to alkaline salt, the genomic DNA methylation levels went up rapidly and reached the maximum in the Na2CO3 treatment. From the result of DNA methylation status analysis, we could learn that the methylated band ratio (% polymorphic bands) in leaves of Zhong9806 was 40.00% and 50.00%, respectively, and demethylated band ratio (% polymorphic bands) was 54.12% and 46.67%, respectively. The methylated band ratio (% polymorphic bands) in roots of Zhong9806 was 35.53% and 43.59%, respectively, and demethylated band ratio (% polymorphic bands) was 56.58% and 51.28%, respectively. But the methylated band ratio (% polymorphic bands) and the demethylated band ratio (% polymorphic bands) in leaves and roots of ZhongS9612 changed a little after stress. In total, 6 fragments were obtained from the gel, which were amplified and analyzed with BLAST method. The result indicated that the 6 sequences were homologous to 6 different genes, widely distributed in coding region and non-coding region, involving different metabolic reactions. The analysis of qRT-PCR showed that these genes were differentially expressed between control and treatment.【Conclusion】The response of cotton varieties with different salt-tolerances to the stress of different salt is different. The DNA methylation level of Zhong 9806, a salt-tolerant variety, declined under salt stress, which could induce the expression of genes related to salt-tolerance, but Zhong S9612, a salt-sensitive variety, lack relevant genes to make the damage increase, reaching the most after the Na2CO3 treatment. A big difference exists in the DNA methylation level between the control and treatment，and the pattern of DNA methylation has tissue specificity. From the analysis of polymorphic fragments, homogenous genes involve many metabolic pathways, through the synergistic effect of three pathways to deal with the stress.

The Possible Effects of Global Warming on Cropping Systems in China Ⅹ. The Possible Impacts of Climate Change on Climatic Suitability of Spring Maize in the Three Provinces of Northeast China

ZHAO Jin, YANG Xiao-guang, LIU Zhi-Juan, LV Shuo, WANG Jing, CHEN Fu

Scientia Agricultura Sinica. 2014, 47(16): 3143-3156. doi:10.3864/j.issn.0578-1752.2014.16.003

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【Objective】The variabilities of distribution patterns of spring maize climatic suitability in the three provinces of Northeast China under climate change were studied, and the results will provide a scientific basis for spring maize cropping distribution in the region. 【Method】In this research, the year 1981 was taken as a time node and divided the period 1961-2010 into two sub-periods. The historical climate data from 74 meteorological stations in these three provinces from 1961 to 2010 were used. According to the agro-meteorological indices, the possible northern limit changes of spring maize in Northeast China were analyzed. The rain-fed yields in each station in the potential cropping region of spring maize were assessed by APSIM-Maize model year by year. On the basis of statistical methods, the variabilities of the rain-fed yield level and stablity zones were investigated. In summary, the variability of distribution patterns of spring maize climatic suitability were studied. 【Result】In the period from 1981 to 2010, the possible northern limit of spring maize moved with a northwestward shift of 158.3-285.8 km, and the possible planting area increased by 3.87×104 km2 (4.91% of the whole land area in Northeast China) than during the period from 1961 to 1980. During the period 1981-2010, the percentage of the very high, high and moderately high rain-fed yield zones of spring maize in the possible planting area increased from 81.14% to 86.66%, but the percentage of the very high, high zones decreased from 36.61% to 34.82% and the moderately high zone increased from 44.53% to 51.85%. Meanwhile, the percentage of the marginally high zone decreased from 18.86% to 13.34%. Generally, the rain-fed yield per unit area reduced by 40 kg•hm-2 in the research area. However, the total rain-fed yield increased by 7.0% with possible planting area increase, especially the high and moderately high rain-fed yield zones. The percentage of the very stable, stable and moderately stable rain-fed yield zones of spring maize in the possible planting area increased from 80.20% to 89.28%. Meanwhile, the percentage of the very stable and stable rain-fed yield zones increased from 40.97% to 49.97%, but the percentage of the marginally stable rain-fed yield zone decreased from 19.80% to 10.72%. The percentage of the suitability and moderately suitable climatic zones increased from 61.09% to 83.00%, but the percentage of the very suitable climatic zone decreased from 18.83% to 6.67%. Meanwhile, the percentage of marginally suitability zone decreased from 20.08% to 10.33%. The rain-fed yield per unit area obtained stably reduced by 171 kg•hm-2 in the research region. However, the total rain-fed yield obtained stably increased by 2.6% with possible planting area increase, especially the suitable and moderately suitable climatic zones. 【Conclusion】Under the background of climate change, the possible northern limit of spring maize moved northwestward with an increasing possible planting area in Northeast China. If there were no changes of varieties, cultivation and management, the percentage of the very high rain-fed yield zones in the possible planting area decreased and the yield per unit area reduced. However, the total rain-fed yield increased with the possible planting area increase. The stability of rain-fed yield increased with a greater percentage of the very stable and stable rain-fed yield zones in the possible planting area increased. In the potential cropping area of spring maize in Northeast China, the percentage of the climatic suitability and moderately suitability zones increased, but the percentage of the very suitable climatic zone decreased. The rain-fed yield per unit area obtained stably reduced. However, the total rain-fed yield obtained stably increased with the possible planting area increase in the research region, especially the suitable and moderately suitable climatic zones.

Climate Change and Agricultural Production in Africa: A Review of Impacts and Adaptations

SHI Wen-Jiao, TAO Fu-Lu

Scientia Agricultura Sinica. 2014, 47(16): 3157-3166. doi:10.3864/j.issn.0578-1752.2014.16.004

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Climate change has a negative impact on agriculture and food security, and this implication will be further going on and on in the future. We need to improve our understanding of the effects of climate change on agricultural production in Africa, and to know the responses of climate change to African agriculture. These are critical keys to adapt to climate change for agriculture in time. This paper introduced the response and adaptation of African agriculture to climate change, and summarized that statistical model, crop model and economic model are the three tools for researching this issue. The responses of climate change to African agriculture in the past and future were reviewed. The conclusions for possible effects of climate change on African agriculture in the future were different, depended on different time and space scales, research methods and crop types. The ranges of the effects were from -84% to 62%, -57% to 30% and -100% to 168% assessed by crop models, statistical models and economic models, respectively. As the big effects of climate change on African agriculture, the adaptation of African agriculture to climate change has been studied by more and more researchers. Adaptation measures like changing cultivars, conservation agriculture, irrigation and adjusting technology will bring more benefits for food security. In addition, some other adaptations, like strengthening the monitoring and warning of climate change, enhancing climatic forecasting, planning crop growth and manage measures using climatic information, adjusting crop allocation, playing advantages of local and international organizations, can also improve the adaptation ability of African agriculture to climate change. The uncertainties, including data, method, results, the indirectly effects of climate change and the lacking of integrated researches, were also discussed. Finally, the future work in this issue was presented. This study will help people to better understand the responses and adaptations of African agriculture to climate change, to better solve food insecurity and to reduce poverty. In addition, it can also provide effective information for the adaptation of agriculture to climate change in China.

Mechanism Analysis of Kynurenine 3-monooxygenase Gene BcKMO in Regulation of Pathogenicity in Botrytis cinerea

LI Pei-Fen, ZHAO Fu-Xin, DONG Li-Ping, ZHENG Hui-Xin, ZHAO Bin, HAN Jian-Min, XING Ji-Hong, DONG Jin-Gao

Scientia Agricultura Sinica. 2014, 47(16): 3167-3173. doi:10.3864/j.issn.0578-1752.2014.16.005

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【Objective】The objective of this study is to reveal the molecular mechanism of kynurenine 3-monooxygenase gene BcKMO in regulating pathogenicity of Botrytis cinerea and lay a foundation for clarifing the pathogenic mechanism of B. cinerea in the future.【Method】The activities of polymethylgalacturonase (PMG), endopolygalacturonase (PG), cellulase (CX), polygalacturonic acid transeliminase (PGTE) and pectin methyl transelimination enzyme (PMTE) were analyzed in the wild-type strain (WT), the BcKMO gene ATMT mutant (BCG183) and gene complement mutant (BCG183/BcKMO) by DNS and Hoffman methods. The toxin was isolated from WT, BCG183 and BCG183/BcKMO and the activity of toxin was analyzed. Acid production assays was performed on PDA medium with bromothymol blue. Penetrability of WT, BCG183 and BCG183/BcKMO was detected with onion epidermis spread on PDA medium. Real-time PCR was used to measure the transcription levels of pathogenicity-related genes, e.g., Bac, Bcg2, Bcg3, PkaR, Bmp1, Sak1, Bcreg1, Bos1, Bcp1, Ras2, Bcpg1, and Sod1 in WT, BCG183 and BCG183/BcKMO.【Result】The activity of PMG and PG in mutant BCG183 were significantly higher than that of WT and BCG183/BcKMO. The CX, PGTE and PMTE activities were no significant difference while compared with WT and BCG183/BcKMO. The toxin activity of mutant BCG183 was significantly higher than that of WT and BCG183/BcKMO. The acid production of the mutant BCG183 significantly decreased. The penetrability of mutant BCG183 appeared no significant difference compared to WT and BCG183/BcKMO. The expression level of pathogenicity-related genes, i.e., Bac, Bcg2, Bcg3, PkaR, Bmp1, Sak1, Bcreg1, Bos1, Bcp1, Ras2, Bcpg1, and Sod1, was obviously up-regulated in mutant BCG183.【Conclusion】The BcKMO gene is involved in regulating cell wall degradation enzyme activity, toxin activity, acid production, penetrability, and the expression of pathogenicity- related genes in B. cinerea.

Cloning of a Polyphenol Oxidase Gene (GhPPO1) of Gossypium hirsutum and Its Role in Cotton after Helicoverpa armigera Feeding

ZHU Xiang-Zhen, MA Qiao-Ying, ZHANG Shuai, 吕Li-Min , LUO Jun-Yu, WANG Chun-Yi, CUI Jin-Jie

Scientia Agricultura Sinica. 2014, 47(16): 3174-3183. doi:10.3864/j.issn.0578-1752.2014.16.006

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【Objective】 The objective of this study is to clone and characterize GhPPO1 from cotton (Gossypium hirsutum), then to study the dynamic changes of GhPPO1 mRNA expression level and PPO activity induced by Helicoverpa armigera in order to clarify the function of this gene in defensing response in cotton. 【Method】 A fragment of PPO gene which has an obvious response to insect feeding from cotton SSH library was got. Specific primers were designed to conduct the 5′RACE reaction. After sequencing and assembly, homologous retrieval was carried out in the NCBI cotton dbEST database and an EST (GenBank accession number: DR461072.1) which have a 410 bp overlap with targeted PPO gene were found. After assembly and electronic extension by DNAstar software, the complete sequences of cotton PPO gene, named GhPPO1, was got. To verify the sequence authenticity of GhPPO1, primers were designed at the two ends of the gene. Using genomic DNA as template, PCR was done to check whether there exist introns in GhPPO1. After verification, the tool of BLASTX was used to analyze the sequence homologous and ClustalW software was used to do multiple sequence alignment and MEGA 4.0 was used to construct a phylogenetic tree and other softwares including ANTHEPRO5.0, ExPASy and InterProscan were used to predict the existence of functional sites and physicochemical properties in the speculated coding regions. The GhPPO1 mRNA expression level and PPO activity in cotton leaves were detected by real-time quantitative PCR and spectrophotometric method, respectively. 【Result】 The complete cDNA sequence of GhPPO1 is 2 022 bp, with a 1 797 bp open reading frame which encodes 598 amino acids (M= 67.18 kD and pI= 6.11). The 5′ and 3′ untranslated regions were 102 and 123 bp, respectively. There were no introns in this gene. In the amino acid coding sequence, there were CuA and CuB binding sites, 3 N-glycosylation sites, 8 N-nutmeg acylation sites, 6 protein kinase C phosphorylation sites, 8 casein kinase Ⅱ phosphorylation sites, 1 cAMP and cGMP dependent protein kinase phosphorylation site and 1 amidation site. In the CuA and CuB binding regions, there existed the histidine and cysteine which play a key role in PPO protein. The similarity of PPO protein between cotton and other plants were more than 50%, however, the relationship of GhPPO1 protein was relatively far away with other kinds of PPO proteins. After mechanical damage, cotton leaf GhPPO1 expression showed a trend of rise and fall. The expression level was the highest at 6 h and the maximum fold change was 3.29 times compared with un-treated leaves. GhPPO1 mRNA level showed a variable trend which was increased firstly and then decreased and then increased at last after 1st late instar cotton bollworm larvae feeding. Twelve hours after feeding, the expression reached the highest level which was 6.04 times higher than the un-treated leaves. However, the mRNA expression level was lower in cotton bollworm feeding leaves than in mechanically injured ones at both 3 h and 6 h. PPO activity showed an increasing trend after both insect feeding and mechanical wounding treatment. The enzyme activity was lower in former treatment. Compared with the untreated cotton leaves, the mRNA expression level and enzyme activity significantly up-regulated after mechanical injury and water jointly treated leaves. However, there were no significant difference between the oral secretion treated and untreated cotton leaves in both GhPPO1 mRNA expression level and PPO activity. It indicated that oral secretion from cotton bollworm larvae might have a suppression role in both GhPPO1 mRNA expression and enzyme activity.【Conclusion】The full-length cDNA sequence of GhPPO1 which plays an important role in defensing cotton bollworm was cloned from cotton. It is speculated that some inhibitors may exist in the oral secretion of cotton bollworm larvae, which may cope with the defense system induced by insects.

Biochemical and Molecular Characteristics of Glutamic Decarboxylase from Bactrocera dorsalis

WEI Dong, WANG Tao, DOU Wei, WANG Jin-Jun

Scientia Agricultura Sinica. 2014, 47(16): 3184-3194. doi:10.3864/j.issn.0578-1752.2014.16.007

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【Objective】 The study aimed to determine the γ-aminobutyric acid (GABA) content and glutamic acid decarboxylase (GAD) specific activity of Bactrocera dorsalis, and clone the complete sequence of a GAD gene (BdGAD1). The changes of GABA content, GAD specific activity, and expression of BdGAD1 in different developmental stages and body tagmata of adults after avermectin stimuli provided basic data of the resistance mechanism of avermectin mediated by GABA. 【Method】 The content of GABA in B. dorsalis was determined through the method of high performance liquid chromatography, and the dose and time effects of avermectin stimuli on GABA content were determined. The change of specific activity of GAD in B. dorsalis was determined via the microplate method with the substrate of glutamate. According to the screened GAD gene sequence fragment from the transcriptome data of B. dorsalis, the complete sequence of cDNA was amplified using rapid amplification of cDNA ends (RACE). The open reading frame (ORF), deduced amino acid sequence, and molecular weight were predicted, and a phylogenetic tree with GAD genes from other insects was constructed using maximum likelihood method to clarify its phylogenetic relationship. Besides, the RNA was extracted from different developmental stages (egg, larva, pupa, and adult) and different tagmata (head, thorax, and abdomen) of adult. Based on the reference evaluation, α-Tubulin was used as housekeeping gene for qPCR to analyze the expression profiles of different developmental stages, tagmata, and stimulated by avermectin. 【Result】 The GABA contents of B. dorsalis increased under the stimuli of avermectin, and there was a positive correlation between GABA content and the avermectin dose and treatment duration, suggesting that B. dorsalis may mediate the content of GABA to avoid the damage of avermectin. Moreover, the specific activity of GAD in B. dorsalis also increased with the increase of treatment doses. A complete sequence of BdGAD1 was cloned by RACE amplification with a full length of 1 755 bp, and ORF of 1 197 bp encoding 398 amino acids. The GenBank accession number was KC763804. This gene exhibited a close relationship with the gene from Anopheles gambiae based on maximum likelihood phylogenetic tree. The amino acid identity was up to 97%. The expression level of BdGAD1 was the highest in larva among different developmental stages, and was the highest in abdomen among different tagmata. Under the stimuli of avermectin, the expression of BdGAD1 was also unregulated.【Conclusion】 Avermectin could increase GABA content by increasing the expression level of BdGAD1 and specific activity of GAD resulting in more GABA synthesis. This might be one reason for the resistance of B. dorsalis against avermectin.

Development of China Digital Soil Maps (CDSM) at 1﹕50 000 Scale

ZHANG Wei-li, ZHANG Ren-lian, XU Ai-guo, TIAN You-guo, YAO Zheng, DUAN Zong-yan

Scientia Agricultura Sinica. 2014, 47(16): 3195-3213. doi:10.3864/j.issn.0578-1752.2014.16.008

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【Objective】High priority has been paid on development of soil geo data base based on soil legacy data by international scientific community in recent years. Temporal and spatial soil information with high resolution, as a new and indispensable tool for researchers and managers, can be applied to soil and environmental quality assessment, arable land conversation, flood and draught mitigation, erosion control, non-point source pollution control, soil and agricultural product pollution control and many other areas. In last decades, several nation-wide soil surveys have been conducted in China. Large number of soil data has been obtained. However, the valuable soil survey data, still in hand drafting map or paper printing format, has been unavailable to researchers and public shares, as the data have been stored at different locations and have not been processed, digitalized and integrated. The purpose of the study is to develop China Digital Soil Maps (CDSM) through extracting soil information from legacy data collected from the whole country. 【Method】Data model of CDSM was constructed by analyzing the characteristics of soil legacy data from different time periods and with consideration of future trends to topics of soil survey. The data model confined the kinds and scopes of information to be extracted and compiled for CDSM. A data treatment working flow was developed, that composed of about 180 separated processes for extracting, analyzing and integrating heterogeneous and massive soil information of different regions and periods. CDSM has been completed by using the methodology for processing massive soil geo data and a computer software program IMAT (Intelligent Mapping Tools) developed in the study. 【Result】CDSM was mainly composed of two kinds of soil geo information. One kind was vector maps of the whole country at 1﹕50 000 scale with polygon objects to describe soil types, soil organic matter, total nitrogen, available phosphorus, available potassium and pH-value. The second kind was point data of about 100 000 soil profiles and 80 000 plough layer samplings with description of sampling point coordinates, sampling year, and soil physical and chemical properties obtained from soil survey works since 1980s. The information contains both the slow-changing soil features such as soil types, parent material, soil mechanical composition, texture and other morphological features and the fast-changing properties such as concentrations of soil carbon and nutrient concentrations. 【Conclusion】The study shows that the CDSM data model is fit both to accept different kind of legacy data from soil surveys across the country over 30 years and to further extend and expand in future. With the S90PT point layer, information of soil sampling points from the new round soil survey works can be added into CDSM. According to time series, space sequence or thematic order, required soil information of the whole country with a map resolution of 100 m×100 m can be inquired and provided by CDSM. The information has been applied by a number of agricultural, environmental and land resource organizations, divisions and research institutes for knowing current soil quality status and understanding changes in soil and environment quality. Along with the extension in time, space and themes in the future, CDSM spatial and temporal soil information will have broader application in various fields.

Review of Soil Classification and Revision of China Soil Classification System

ZHANG Wei-Li, XU Ai-Guo, ZHANG Ren-Lian, JI Hong-Jie

Scientia Agricultura Sinica. 2014, 47(16): 3214-3230. doi:10.3864/j.issn.0578-1752.2014.16.009

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During China Second Soil Survey, a soil classification system developed by hundreds of soil scientists was used as the normative standard in the survey. Since then, the system has been recommended and applied as the national standard. The first integration of county data from the survey showed that there were differences between the soil names extracted from county data and the names of the national system. In order to revise soil names which did not match with the national classification system, to clarify the differences among the two existing soil classification systems in China at present and the system recommended in World Reference Base for Soil Resources (WRB), research works on soil classification in last decades were reviewed and discussed in this paper.It was showed in the study that soil genesis has been applied as the common basis for soil classification in different countries. Although the theoretical basis is the same, different countries developed different soil classification systems, as the concepts, the naming conventions, the ground survey methods and the soil sampling density are different in different countries. Main reasons for that were large differences in climatic zones, richness of soil types, levels of economic and technological development at different continents and countries. Because of the language custom, the influences of already existing classification systems and the declining demand in understanding pedogenesis in soil surveys in recent years, the impact of efforts made for integrating different soil classification systems has not been obvious.Comparison among soil classification systems in different languages has been showed that the officially issued national soil genesis classification system is more in line with Chinese language characteristics. Main features of the main soil types of China are well extracted and expressed with names of the 60 Soil Great Groups of the system. These names have been approved to be easy for both the soil classification experts and the soil scientists to distinguish and cognize the dominating soil formation processes, the main futures and the spatial distributions of main soil types of the country. Since the system has been officially recommended and applied for more than 30 years and had a great impact on country-wide, so the authors suggest that the system should be used continually. Problems of the classification system, in regard with mismatching with names of soil taxonomy of WRB, should be solved by establishing the relationships between 60 Soil Great Groups of China and the soil names of WRB. The review showed that it would be conducive to the stability of the classification system and the cognition of major forming process of soil types by soil scientists, if the naming of soil types were confined to soil formation. Changing classification system in the hierarchy or introducing parameters besides soil formation process into the system may lead to burdensome soil names, weakening the cognition of soil formation process. On the other hand, information of soil genesis and classification can supply crucial supplementary information to evaluate and understand soil functionalities such as soil fertility, environment and health properties. According to the points mentioned above, soil names extracted from county data in the survey were revised and summarized. According to China National Classification System, the soil great groups were merged to 60. The names of soil sub groups were moderately merged. Names of soil genus and species, which contained rich soil forming information from survey works, were maintained as far as possible.

A Summary of Methodology for Extracting, Integrating and Mapping of Massive Geo-Data

ZHANG Wei-Li

Scientia Agricultura Sinica. 2014, 47(16): 3231-3249. doi:10.3864/j.issn.0578-1752.2014.16.010

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The methodology for extracting, integrating and mapping of massive geo-data is a new method combined by different disciplinary approaches in agriculture and environmental science, geo science, cartography, information and computer science. The methodology consists mainly in the data model design and the working flow design for processing, analyzing and mapping massive geo-info. By using the methodology, big data in heterogeneous format and structure originated from different resources and regions of agricultural and environmental research and working programs can be effectively extracted, analyzed and correlated for mapping and thematic expression. The method can be used to analyze point observation data, map data, remote sensing data in different spaces and times. So that it supplies a useful tool for providing information with higher accuracy, larger covering area and more system concerning elements, through which major factors and mechanism in agricultural and environmental system can be much more exactly qualified and quantified. Based on many years of research and practical experience, the author of this paper introduced the connotation, the application range, the basic and the concerning conceptions and the main contents of the methodology, with the purpose to provide a brief understanding for researchers and managers in agriculture and the environment sectors to apply the new method in their working fields. As a big data approach, the method can be widely used for evaluation of the quantity and quality of soil resources, climate change, environmental quality, evaluation of crop varieties suitable distribution area, agricultural non-point source pollution control，erosion control, drought and flood disaster mitigation, and other working and research areas. It can also be applied for precise and visualized expressing of soil fertility and environmental quality, so that farmers or other users from different sectors can access to research results and progress much easier and get benefits from it. It provides also a useful approach to establish scientific basis for developing and implementing incentive policy in agriculture and environment sectors. The core of methodology is to define the rules according to scientific target for classifying massive geo-data. Based on the rules defined, grouping, coding, extracting and mapping of massive geo-data can be then carried out. Because of horizontal and vertical features of data structure of a massive geo-dataset, the four component expression of massive geo-dataset should be applied. Through which both logical and physical structure differences of massive geo information originated from different sources can be distinguished and displayed clearly. After normalization of data logical structure, the extracting, integrating and mapping of massive geo-datasets are then followed. In agriculture and environmental research works, however, frequent difficulty of big data analysis approach is the weakening or even lost of the scientific target during data treatment. Therefore, scientific or specific target should be defined as precise as possible at the beginning of the data analysis working program. A five-level designing process should be applied for drafting working flow of data extracting, integrating and mapping. Checking and examining the realization of defined target should be done time to time during the data processing. With deep understanding of the target, researchers and professionals from agriculture and environment sectors should be responsible for designing data processing flow of high-levels and drafting the corresponding design documents according to specifications of the methodology.

Design and Development of Software Package Intelligent Mapping Tools (IMAT)

ZHANG Wei-Li

Scientia Agricultura Sinica. 2014, 47(16): 3250-3263. doi:10.3864/j.issn.0578-1752.2014.16.011

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【Objective】In agricultural and environment research area, restrained by financial and technical limitations, in fact, most studies have to focus on specific topics and mechanisms based on point or parcel observations and confined period. As a result, large number of scattered observation data was produced. Different types of observations and evidences previously obtained in separate studies can be related through associated coordinates. Such connections may induce new scientific acquaintance from former observations and evidences. Massive geo-data analysis methods improve the comprehending of process or object complex through multi- channels and with multi-point of view. It is a useful tool to examine and amend the assumptions to the world by taking advantage of the new data and evidence. The main difficulty of the big geo-data analysis comes from not only the treating of large data volume, but also the extracting, integrating and expressing information based on heterogeneous data resources. Since there has been no mainstream software tool available for big geo-data analysis, purpose of the study is to develop a professional tool for extracting and integrating heterologous massive geo-information from different resources as well as for making thematic maps with high resolution on large-scales. The intelligent software package should finish data processing and mapping automatically or human-computer interactively.【Method】Principles and rules of methodology for massive geo-data analysis and software design were applied for IMAT (Intelligent mapping tools) design. The whole design consisted of three parts, the system architecture, the system data supporting platform, and the design for system modules and models. For IMAT software development, C# was used as the programming language, NET Framework 4 Extended was applied as development environment, functions and components from software packages of ArcGIS, Access and DotNet Bar were called. 【Result】With 38 independent functional modules, IMAT provides the main functions for analyzing massive spatial information and cartographic representing, which are required in agriculture and the environment research and working area. Each module can be used to independently conduct certain data analysis and processing, for example, the big data loading and storing, the statistical analysis, classification and coding of space elements, the data selecting, integration and mapping, etc. It can also be used as a combination of several modules to complete a more complex task of data extraction and expression. IMAT has made up the lack in functions to deal with massive spatial data provided by mainstream database package and GIS software package. The objects for IMAT data analysis are the massive spatial database. When doing data analysis with IMAT, rules for data extracting can be set down based on thematic objectives. The integration of massive spatial data within logical data structure as well as storage data structure can be processed automatically or in batch processing way. When making cartographic representations with IMAT, rules and parameters for the whole map composed of a series map sheets can be set, through which differentiated data extracting and mapping for map sheets can be carried out. The data analysis and mapping procedures can be operated by intelligent and automatic way as well as human-computer interactive approach.【Conclusion】For agricultural and environmental topics, IMAT can be applied to analyze and express thematic elements both for soil types and soil texture that expressed by classification, and for soil nutrient and contaminant concentration or non-point source pollution index that expressed by quantity. In addition, it can be used for extracting multi thematic elements and for mapping with different map scales or map sheets. In IMAT design, the whole system was assembled by separated modules and models, so that both the designing and the programming of the software packages could be completed efficiently. By using a four component expressions as interface files among different massive geo-data aggregate from different modules, each module of IMAT is able to receive and process geo-data aggregate of different heterogeneity at different data processing steps. According to data processing target, whether for data extracting, integrating or mapping, required functional modules can be chosen and assembled with high flexibility.

Cloning and Expression Analysis of Fertility-Related Genes for the Genic Male Sterile Line in Pepper (Capsicum annuum L.)

LIU Chen, MA Ning, FU Nan, LI Xin, GUO Shuang, SHEN Huo-Lin

Scientia Agricultura Sinica. 2014, 47(16): 3264-3276. doi:10.3864/j.issn.0578-1752.2014.16.012

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【Objective】To investigate the correlativity between the four screened ESTs and the genic male sterility, full length cDNA of the four genes were obtained, and the expression patterns of different materials and different organs were analyzed.【Method】The expression patterns of the four genes were detected by RT-PCR. Full length cDNA were cloned using the rapid-amplification of cDNA ends technique (RACE). Physical and chemical properties of proteins were analyzed with the bioinformatics softwares. RT-PCR was employed to detect expression patterns of the genes in different organs (anther, ovary, petal, sepal and leaf) and anthers from different developmental stages (tetrad stage, early- or mid-uninucleate stage, late-uninucleate stage and binucleate stage).【Result】According to the results of Blastx, the four genes were named as CaSEP1, CaPROF, CaOle e 6 and CaPCP, separately. Full length of CaSEP1 is 1 108bp, which codes 221 amino acids and contains a MADS domain and a K domain. The full length CaPROF includes 767 base pairs and 131 amino acids, which has a PROF domain. CaOle e 6 is 523 bp in full length, codes 85 amino acids with an Ole e 6 domain. Full length of CaPCP includes 563 base pairs and 66 amino acids. Amino acid sequence alignments and phylogeny reconstructions indicate that CaSEP1 is most similar to the tomato SEP1 protein and they have the closest relationship. CaPROF is more similar to profilins from tobacco and tomato than other species and shows the closest genetic distance with tomato profiling, CaOle e 6 is similar to the tomato Ole e 6 protein and they are classified into the same cluster, CaPCP shows the most similarity with tea pollen like protein and they are closest in genetic distance. RT-PCR showed that CaSEP1 expressed only in the reproductive organs of the fertile line with higher expression in anther, ovary and petal than sepal and leaf. CaPROF expressed significantly higher in anther than leaf of the fertile line, while no expression in other organs. CaOle e 6 expressed significantly higher in anther than other organs of the fertile line. CaPCP showed specific expression in anther of the fertile line. The expression of CaSEP1 increased gradually before decreasing in the binucleate stage of fertile anther, while it increased gradually in sterile anther during microspore development. The gene shows a similar expression between fertile line and sterile line at the tetrad stage of microspore development. At early- or mid-uninucleate stage and late-uninucleate stage, the gene expresses higher in fertile line. While the expression in fertile line is lower than sterile line during binucleate stage of microspore development. CaPROF, CaOle e 6 and CaPCP only express at the late stages (late-uninucleate stage and binucleate stage).【Conclusion】Analysis of sequences and expression profilings of the four genes indicates close relationships to male fertility. This study provides important information for revealing the mechanism of male sterility and regulating male fertility in pepper.

Cloning and Expression Analysis of Glutathione Reductase Genes(CsGRs) in Tea Plant (Camellia sinensis)

YUE Chuan, CAO Hong-li, ZHOU Yan-hua, WANG Lu, HAO Xin-yuan, WANG Xin-chao, YANG Ya-jun

Scientia Agricultura Sinica. 2014, 47(16): 3277-3289. doi:10.3864/j.issn.0578-1752.2014.16.013

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【Objective】 The objectives of the present study were to clone the genes of glutathione reductase family (CsGRs) from tea plant (Camellia sinensis) and investigate their functions under different abiotic stresses. 【Method】 According to the sequences of CsGR genes obtained from the transcriptome database of tea plant, specific primers were designed for reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR) to clone the full-length sequences of CsGRs. The bioinformatic characteristics of the cloned CsGRs were analyzed using online service. The expression profiles of CsGRs in various tissues and in response to cold, drought, salt and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR). Spectrophotometer technique was employed to determine the content of glutathione (GSH) upon cold and drought stress. 【Result】 CsGR1 was isolated from tea plant using RT-PCR and had 1 827 bp in length containing a 1 482 bp open reading frame (ORF) encoded 493 amino acid residues. For CsGR2 isolating, 712 bp and 1 624 bp in length of 5′ and 3′ terminal sequences were amplified by RACE-PCR, respectively, after sequences assembling and verified by RT-PCR, CsGR2 with 2 282 bp in length containing a 1 698 bp ORF encoded polypeptide of 565 amino acids was obtained. CsGR1 and CsGR2 were submitted to GenBank with accession number KF906411 and KF418080, respectively. The molecular weights of CsGR1 and CsGR2 encoded protein were 53.9 and 61.0 kD, respectively, and both of them did not contain the signal peptide sites, indicating that they were not the secretory proteins. Subcellular localization prediction showed that CsGR1 might localize in cytoplasm without chloroplast transit peptides (cTP), whereas CsGR2, containing a putative cTP of 71 amino acid residues at the N-terminal, was most likely to target to chloroplast. Comparison of sequences similarity with reported GRs showed that CsGR1 had more than 80% similarity with cytosolic-GRs and less than 60% similarity with chloroplastic-GRs, whereas CsGR2 shared over 70% identity with other chloroplastic-GRs and about 50% similarity with cytosolic-GRs. CsGR1 and CsGR2 shared 63.4% and 49.9% sequence identity in nucleotide and amino acid, respectively, and had high similarity with each other in secondary structure of protein. Phylogenetic tree analysis showed that CsGR1 and CsGR2 could be clustered into cytosolic-GRs and chloroplastic-GRs, respectively, and they had the closest genetic relationship with Vitis vinifera. The redox-active disulfide bridge, the glutathione-binding residues and the conserved arginine residues for NADPH binding were found in both of them. Hence, CsGR1 encodes a putative cytosolic isoform, and CsGR2 belongs to chloroplastic GR which dual-targeted to both chloroplasts and mitochondria. Transcript abundance of CsGR1 was higher in flowers and roots than that in leaves and stems. And CsGR2 displayed the opposite expression pattern in tissues compared to CsGR1. Analysis of the expression patterns in response to abiotic stress revealed that CsGR1 and CsGR2 were down-regulated by ABA treatement in leaves and the suppression level of CsGR2 was higher than CsGR1. Furthermore, the expression of CsGR1 was repressed by cold (4℃) stress, whereas CsGR2 could be gradually induced with the extension of treatment time. Similarly, under salt stress, the expression of CsGR1 was suppressed, and CsGR2 was up-regulated after 24 h treatment. Under PEG treatment, both of CsGRs were up-regulated in leaves and down-regulated in roots. Analysis of the concentration of GSH showed that GSH content was gradually induced by cold and PEG treatment in leaves. Moreover, the GSH concentration could be returned to and kept at constant level after 48 h recovery in PEG treatment. 【Conclusion】 In this study, two CsGRs were cloned from tea plant, and their expressions were regulated by ABA, cold (4℃), salt and PEG stress. The results demonstrated that GR might play a role in abiotic stresses tolerance of tea plant.

Accumulation of Reactive Oxygen Species Related to Disease Resistance Induced by BABA in Postharvest Banana (Musa AAA. cv. Brazil) Fruit

TAN Wei-ping, PANG Xue-qun, ZHANG Zhao-qi, HUANG Xue-mei

Scientia Agricultura Sinica. 2014, 47(16): 3290-3299. doi:10.3864/j.issn.0578-1752.2014.16.014

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【Objective】 Effects of β-aminobutyric acid (BABA) on diseases-tolerance of harvested banana (Musa AAA Group cv. Brazil) and the possible mechanism were investigated to provide theoretical basis for the new technology of resistant and preservation. 【Method】 In this study, diphenylene iodonium (DPI), a NADPH oxidase specific inhibitor, was used to inhibit ROS generation in BABA treated fruits. After 5 g•L-1 BABA treatment or 3.14 mg•L-1DPI followed with 5 g•L-1BABA treatment, banana fruits were inoculated with Collectotrichum musae (2 ? 105 spores/mL) at 0, 3, 6, 12, 24, 48, and 72 h, respectively, and stored at 20±2℃ and RH 85%-95%. The disease spot sizes were determined at 5 d to 16 d. The fruit inoculated with C. musae at 24 h after BABA or DPI treatments and stored at (20±2)℃ and RH85%-95%, and the peel was used for determination of production rate, activities of catalase (CAT) and ascorbate peroxidase (APX), β-1, 3-glucanase(GUN), phenylalanine ammonia lyase(PAL), chitinase (CHI), gene expressions of MaCAT, MaAPX, MaGLU, MaCHI, MaPAL1 and MaNOX during storage. 【Result】 BABA treatment effectively reduced the disease spot sizes on the peel of inoculated banana at 24 h after BABA treatment and cultured for 5 d, showing that BABA treatment needed proper time to have an effect. The production rate and MaNOX gene expression of BABA treated fruits were evidently higher than those of control during 5-12 d storage. Compared with the control, CAT activity and MaCAT expression were higher on day 5 and 1-5 d, respectively. APX activity and MaAPX expression were higher on 5-8 d and 14 d, and 1-5 d, respectively. CHI activity and MaCHI expression was higher on 5-12 d and 1-5 d, respectively. Both of GUN activity and MaGLU expression were higher on 8-14 d. Both PAL activity and MaPAL1 gene expression were higher on 12-14 d . No significant difference was found in other time points. DPI combined with BABA treatment suppressed the above effects of BABA treatment.【Conclusion】These results strongly suggest that reactive oxygen species was involved in the BABA-induced disease tolerance. BABA treatment started the protection mechanism of reactive oxygen species in banana fruits, including the production of reactive oxygen, the increasing of free radical scavenging enzyme activities, and the response of defense-related gene expression.

Effect of Momordica charantia Fruit Aqueous Extract on Serum Lipids Metabolic Disorder in Restraint Mice

TANG Qin, DENG Yuan-yuan, ZHANG Rui-fen, ZHANG Yan, ZHANG Ming-wei, WEI Zhen-cheng, TANG Xiao-jun, LIU Lei, TI Hui-hui, MA Yong-xuan

Scientia Agricultura Sinica. 2014, 47(16): 3300-3307. doi:10.3864/j.issn.0578-1752.2014.16.015

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【Objective】The objective of this experiment is to study the effect of Momordica charantia fruit aqueous extract（MCFE） on lipids metabolism and oxidative situation in restraint mice and provide the theoretical basis for revealing the improving effect of M. charantia on lipids metabolism disorder.【Method】The 6 weeks old female mice were randomly divided into 5 groups including normal control group (NC) , stress control group (Mod), three MCFE groups (250 mg•kg-1, 500 mg•kg-1 and 750 mg•kg-1), which were administered samples once a day successively for 7 days. After intravenously injected 10% intralipids into the 20 h restrained mice, the content of serum lipid, lipase of mesenteric adipose tissue (MAT) and lipoprotein lipase (LPL) activity of different tissues were determined. The oxidative situation and the antioxidant capacity of the body were measured. 【Result】 The content of TG in Mod was 1.27 times of NC, but the content of low, medium and high MCFE groups were 0.63, 0.57, 0.55 times of NC. MCFE accelerated the elimination rate of TG（P<0.05）in a dose-dependent manner, and improved the fatty acid metabolism. Lipase activity in Mod was 0.77 times of NC, and low, medium and high MCFE groups were 0.77, 0.98, 1.06 times of NC. Lipase activity of medium and high MCFE groups basically reached normal level. MCFE improved lipase activity of mesenteric adipose tissue（MAT）. Compared with the Mod, the LPL activity of serum and MAT in each MCFE group increased significantly with dose-dependent（P<0.05）. But there was no significant effect on muscle LPL activity. The content of MDA and ORAC in Mod was 1.37 and 0.65 times of NC, respectively. The content of MDA of MCFE groups was lower than Mod significantly and the medium, high MCFE groups reached normal. The ORAC of MCFE groups was higher than Mod（P <0.05）. MCFE increased the antioxidant capacity of serum（ORAC）.【Conclusion】 MCFE improved the lipid metabolic dysfunction in restraint mice. The mechanism of M. charantia improves the lipid metabolic dysfunction in restraint mice may be the amelioration of oxidative situation in body and improving the LPL and LIPASE activities of related tissues.

Advances in Differentiation of iPS Cells into Male Germ Cells

DU Jun-Hui, CAO Wen-Guang

Scientia Agricultura Sinica. 2014, 47(16): 3308-3314. doi:10.3864/j.issn.0578-1752.2014.16.016

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Induced pluripotent stem cells (iPS cells) refers to reprograming animals or human somatic cells by gene transfer technology, i.e. using so-called packed viral vectors or particles to infect the cells to be induced. Different somatic cells can be induced into iPS cells through different vectors or different transcription factors combination, such as fibroblasts, hepatocytes, keratinocytes and cord blood, etc. As a new member, iPS cells were similar to embryonic stem cells, i.e. clonal morphology, gene expression pattern, surface marker, embryoid bodies formation, differentiative capacity, teratoma and chimeras (in mice) and so on. Just like ESCs, studies have shown that iPS cells can be induced into different cells in vitro under the conditions of specific induction, including myocardial cells, blood cells and germ cells. It shortens the distance between stem cells and clinical disease, Therefore iPS cells have become the most promising seed cells in cell therapy and tissue organ regeneration, and they have a potential value in the cells’ alternative treatment, pathogenesis research and new drug screening simultaneously. Male sterility not only affects human normal life, but is also very adverse to the development of animal husbandry. Great progress has been made in that iPS cells from humans and mice can be differentiated into primordial germ cells (PGCs), spermatozoa and their precursors. These findings can help not only avoid the difficulties in obtaining ESCs, immune rejection and ethical problems, which may be aroused by using embryonic stem cells, but also provide a good research platform for revealing the developmental mechanism of male germ cells. Patients can use their iPS cells derived male gametes to reproduction. Therefore, differentiation of iPS cells into male germ cells brings new hope for the treatment of male sterility in the future. In addition, differentiation of iPS cells in vitro has broad application potentials for modern animal husbandry. It can be used to produce the transgenic animals. Here, the authors highlighted the advances in research of the differentiation of iPS cells into male germ cells in vitro and its potential application prospects from the different culture conditions and inducers and the prospects in application of the differentiation of iPS cells.

Constructing Bronchial Arteries Stereoscopic Specimen of Pig by Independent and Combined Casting Methods

HAN Zhi-lei, WANG Xiao-liang, LI Lu, YAN Zhen-long, HUO Jing-ping, WANG Bai-xue, SHI Jun-hong, LIU Ying, HE Yu-qin

Scientia Agricultura Sinica. 2014, 47(16): 3315-3322. doi:10.3864/j.issn.0578-1752.2014.16.017

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【Objective】The objective of this experiment is to improve and optimize the ABS vassal casting technique, establish a casting method for a Stereoscopic specimen of pig bronchial arteries, cast a stereoscopic specimens of pig bronchial arteries, thus providing an experimental way for the study of the branching structure and distribution of pig bronchial arteries in lung.【Method】With independent and combined casting methods of bronchial arteries, independent and combined bronchial artery casting specimens were obtained through improving and optimizing the ABS casting method. 【Result】 A method was established for making casts of the stereoscopic structure of bronchia arteries of a pig. This study proposed the steps and methods for making casts of the porcine independent and combined bronchial arteries. The steps and methods for making independent casts of the bronchial arteries were as follows: Firstly, the thoracic aorta was ligated and intubated in the proximal and distal ends of the thoracic aorta. Meanwhile the front end and the rear end of the esophagus were ligated at the corresponding position of the thoracic aorta intubation and the trachea was inserted the blind end sleeve and ligated in order to prevent the leakage around the trachea and esophagus vascular network during the infusion. Secondly, 60 mL of 10% ABS and 100-160 mL of 15% ABS were forced into the aorta through the aorta intubations indirectly. Infusion should be stopped when the middle part of rubber tube dilated. When the dilated part becomes smaller, 20% ABS should be infused to ensure the normal pressure after the end of the continuous infusion 5 h later. Thirdly, these specimens were hardened in water for 3 to 4 d, and then corroded with hydrochloric acid for about 10 d. At last, the integral specimens were obtained though washing under the running water and high pressure, and trimming elaborately. The major points for making combined casting of bronchial artery and bronchial tree were as follows: Firstly, the thoracic aorta, esophagus and trachea were simultaneously intubated and ligated. Secondly, the bronchial artery was infused with indirect aorta intubations. At last the combined casting of bronchial artery and bronchial tree was obtained through the process of hardening, corrosion, washing and repairing. The major points for making combined casting of bronchial artery and pulmonary artery were as follows: The method for making combined casting of bronchial artery and bronchial tree is similar to the method of making combined casting of bronchial artery and pulmonary artery. However, the former and the latter have the differences. For the former, the casting agent is infused into bronchial tree, while pulmonary artery for the latter. The integral independent bronchial artery cast specimens, the combined cast specimens of bronchial artery and bronchial tree and the combined cast specimens of bronchial artery and pulmonary artery were obtained in this study.【Conclusion】The three-dimensional structure of these cast specimens was clear, which could completely display the origin, course, branching and distribution. Meanwhile this model could progressively display the relationships between the bronchial artery and bronchial tree or pulmonary artery. The establishment of the methods could make a good foundation for the study of bronchial artery of pig and other animals.

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