Scientia Agricultura Sinica

Progresses in Research of Physiological and Genetic Mechanisms of Wheat Heat Tolerance

XIN MingMing, PENG HuiRu, NI ZhongFu, YAO YingYin, SUN QiXin

Scientia Agricultura Sinica. 2017, 50(5): 783-791. doi:10.3864/j.issn.0578-1752.2017.05.001

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Wheat (Triticum aestivum) is a second largest staple crop in China, which closely related to the improvement of people’s living standards, and high and stable yield is the most important target in wheat breeding program. Nowadays, heat stress has become one of the limiting factors for wheat production, because wheat is a chimonophilous crop, and very sensitive to heat stress especially at productive stage, which would cause decrease in both wheat quantity and quality. Recently, to analyze the physiological, genetic and molecular basis of heat tolerance in wheat, researchers have developed diverse segregation populations with different heat sensitivities, and mapped dozens of heat-tolerant QTLs on wheat chromosomes in terms of canopy temperature, grain filling duration, membrane stability and chlorophyll content, as well as heat sensitivity index of grain number and thousand kernel weight. In addition, transcriptome, proteome, and epigenome analyses have been applied to identify heat responsive genes, miRNAs and long non-coding RNAs, and some of which have been proved to contribute to the heat tolerance in wheat. Although receptors of heat signal is not yet identified, calcium ion channel and phytohormone e.g. ABA and SA exhibited a crucial role in signal transduction. In this paper, the progresses in research of heat-tolerance related QTL mapping, transcriptome, proteome and epigenome profiling and heat-responsive gene identification were summarized, and proposed that it is necessary to perform systematical evaluation of wheat germplasm, screening of allelic variation and analysis of underlying molecular mechanism for further utilization in wheat breeding program to develop wheat cultivars with high and stable yield in China.

Establishment of an Evaluation System of Shade Tolerance in Soybean and Its Variation in Southern China Germplasm Population

SUN ZuDong, ZHANG ZhiPeng, CAI ZhaoYan, ZENG WeiYing, LAI ZhenGuang, CHEN HuaiZhu, YANG ShouZhen, TANG XiangMin, SU YanZhu, GAI JunYi

Scientia Agricultura Sinica. 2017, 50(5): 792-801. doi:10.3864/j.issn.0578-1752.2017.05.002

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【Objective】 Shade tolerance is an important factor that affects the yield of intercropped soybean, thus limiting the extension of intercropping patterns of soybean. It is necessary to establish an efficient, versatile and reliable shade tolerance identification system to be used in identifying the shade tolerant sources from germplasm populations, which can be used for shade tolerance breeding. 【Method】 Samples of 60 soybean varieties with different shade-tolerance levels were chosen from the germplasm storage in Guangxi Academy of Agricultural Sciences were used in this study. Firstly, the suitable shading treatment for shade tolerance evaluation was chosen among the three shading levels (i.e. 15%, 30% and 60%) according to a reasonable lodging rate of the tested varieties. Secondly, the suitable shade tolerance indicators were chosen from the 17 traits, including plant height, mean internode length, petiole length, shoot fresh weight and so forth. Then, the suitable measuring time was chosen among the three dates, i.e. on the 40th, 50th and 60th day after sowing according to the error coefficient of variation and phenotypic coefficient of variation of the selected shade tolerance indicator. Finally, the shade tolerance evaluation procedure was assembled to include the suitable shading level, shade tolerance indicator and measuring time. Using the designed procedure, the shade tolerance of 453 soybean varieties from southern China was evaluated, from which the tolerant sources were screened out. 【Result】 The 30% shading intension condition was better than other shading degrees. More specifically, there showed lower lodging rate of accessions (22%), higher phenotypic coefficient of variation (25%), and better distinction among accessions. The shade tolerance indicator which composed of the plant height and the average internode length is better than other indicators after comprehensive tradeoff, with advantages as follows: i) It is more accurate due to its smaller error coefficient of variation (9.36%) and higher heritability (95.43%); ii) It is more stable, as its correlation (0.92) between two environments is the largest; iii) It has a better distinction degree, because its phenotypic coefficient of variation (31.25%) and genotypic coefficient of variation (30.52%) are larger; iv) It is more consistent to the field shade tolerance situation with the highest correlation (0.73) between the indicator and visual shade tolerance level. Compared to other measuring times, the shade tolerance indicator on the 50th day after sowing is better, for example its correlation (0.87) between two environments is the highest and error coefficient of variation (7.75%) is the smallest. Therefore, the average of relative plant height and average length of internode was defined on the 50th day after sowing under 30% shading intension condition as the shade tolerance index (STI). The smaller STI, the more shade tolerance of the accession. The STI of 453 soybean varieties from southern China ranged from 1.11 to 2.61, with an average of 1.55 and there showed significant differences among the accessions. In addition, the heritability (91.13%) of these soybean varieties indicated that soybean shade tolerance had high accuracy from phenotypic selection. The STI in eco-region Ⅲ, Ⅳ ,Ⅴ and Ⅵ showed abundant variations with the range of 1.19-2.08, 1.17-2.61, 1.27-2.37, and 1.11-2.54, respectively, which provide materials for the improvement of shade tolerance. Furthermore, the released cultivars and landraces also have abundant variations with the range of 1.11-2.61 and 1.17-2.54, respectively. However, eight of the eleven accessions with high shade tolerance in two environments were released cultivars, which implies the achievement in shade tolerance breeding of soybeans. 【Conclusion】 The shade tolerance indicator which composed of plant height and average internode length on the 50th day after sowing was concluded to be accurate, stable, sensitive and authentic. Using this evaluation system, a number of shade tolerant soybean varieties were screened out from the southern China germplasm population.

Genome-Wide Association Study of the Fertility Restorer Loci for pol CMS in Rapeseed (Brassica napus L.)

WEI DaYong, TAN ChuanDong, CUI YiXin, WU DaoMing, LI JiaNa, MEI JiaQin, QIANWei

Scientia Agricultura Sinica. 2017, 50(5): 802-810. doi:10.3864/j.issn.0578-1752.2017.05.003

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【Objective】 Polima system of cytoplasmic male sterility (pol CMS) in Brassica napus (rapeseed), controlled by a major gene as well as polygenes, has been widely used in China for hybrid rapeseed breeding. Genome-wide association study (GWAS) was performed in a rapeseed population to identify genetic loci and candidate genes for fertility restorer of pol CMS. 【Method】 301A, a rapeseed pol CMS line, was chosen as the female parent to cross with a panel of 308 accessions in natural population of rapeseed which has been genotyped previously using the 60 K Brassica SNP array. The F₁ hybrids were grown for fertility evaluation in 2013 and 2014, respectively, with two replications each year. The fertility of F₁ was classified according to pollen fertility and performance of pistil and stamen, and population structure and relative kinship of 308 male accessions were analyzed. GWAS was conducted by associating the fertility of F₁ with the single nucleotide polymorphisms (SNPs) of males. Candidate genes was identified from the region of 100 kb each side of the peak SNP or trait-associated SNPs at LD (R²＞0.5). Comparative analysis of QTL and haplotype effect evaluation for candidate genes were performed. 【Result】 A significant difference (P＜0.01) was found in the fertility of F₁ between two years, but a high correlation was detected in it between two years (r = 0.52, P＜0.001). The population structure analysis classified the 308 male accessions into three genetic groups (winter, spring and semi-winter). The relative kinships analysis found that 73% of the kinship coefficients between lines were ＜0.1 and 53% were equal to 0. A total of 13 SNPs were detected to be with significant association with the fertility of F₁, formed six genetic intervals on chromosomes A01, A09, C03, C06 and C08. Six genes related to fertility were predicted from the six intervals, and four of these could encode the PPR type proteins which is a conserve structure encoded by fertility restorer genes. Collinearity analysis revealed two PPR type candidate genes (BnaA09g46700D and BnaC08g40710D) detected in homoeologous regions between chromosome A09 and C08 were homology with ORF2, an open reading frame functioning as the reported rapeseed nuclear restorer gene of pol CMS. The other two PPR type candidates (BnaC03g45840D and BnaC06g13000D) were novel candidate restorer genes for rapeseed pol CMS, of which the linked alleles or haplotypes of SNPs were found to significantly associated with the fertility level of the F₁ (P＜0.001).【Conclusion】The present study identified several fertility restorer genes for pol CMS in rapeseed from both A and C subgenomes. Developing functional markers from the alleles or SNPs linked with the candidate genes will benefit the screening of restorer and maintainer lines in the pol CMS system.

Effects of 24-Brassinolide on the Fertility, Physiological Characteristics and Cell Ultra-Structure of Soybean Under Saline-Alkali Stress

WU Yang, GAO HuiChun, ZHANG BiXian, ZHANG HaiLing, WANG QuanWei, LIU XinLei, LUAN XiaoYan, MA YanSong

Scientia Agricultura Sinica. 2017, 50(5): 811-821. doi:10.3864/j.issn.0578-1752.2017.05.004

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【Objective】The aim of this experiment was to study the effects of exogenous EBR (24-epibrassinolide) on the growth indices, physiological characteristics and cellular ultrastructure of soybean under saline-alkali stress. 【Method】Soybean variety Heinong 44 was employed as the test material, and cultivated in 110 mmol·L^-1nutrient solution saline-alkali stress treatments for 3 d and 7 d to investigate the effects of 1.2 mg·L^-1exogenous EBR on the height and root growth, SOD, POD, and APX three kinds of antioxidant enzyme activities, relative electrical conductivity, superoxide anion (O₂^-) production rate, contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), free proline concentration content, chlorophyll content of soybean leaves and the responses of cellular ultrastructure of soybean leaves and root tips under saline-alkali stress. 【Result】Under the condition of saline-alkali stress dealing with 3 d and 7 d, compared to the control, the three kinds of antioxidant enzyme activities of SOD, POD, and APX, free proline content, relative electrical conductivity, O₂^-production rate, contents of H₂O₂ and MDA all increased, the growth indices and chlorophyll content all decreased; the chloroplast and mitochondria cellular ultrastructure of soybean leaves suffered severe damages. The mitochondria and endoplasmic reticulum cellular ultrastructure of soybean root tips suffered severe damages, and vacuole was ruptured. Application of EBR under saline-alkali stress increased soybean plant height, root length, fresh mass of root by 6.45%, 9.60% and 19.85%, respectively. Application of EBR the activities of SOD, POD, and APX of soybean leaves in 3 d and 7 d treatments increased by 16.92% and 9.68%, 48.85% and 61.44%, 19.05% and 20.36%, respectively, relative electrical conductivity, O₂^-production rate, H₂O₂ and MDA contents significantly decreased by 19.58% and 28.26%, 28.06% and 40.92%, 28.62% and 31.21%, 31.03% and 37.17%, respectively, free proline content and chlorophyll content significantly increased by 3.67% and 15.96%, 13.34% and 16.87%, respectively. At the same time, the stability of soybean leaves and root tip cell ultrastructure were maintained and the aging of cells and disintegration were delayed.【Conclusion】The results suggested that application of exogenous EBR under saline-alkali stress could increase the antioxidant enzyme activities, free proline concentration and chlorophyll content, reduce the accumulation of reactive oxygen species(ROS), maintain the cell structure integrity, promote seedling growth and resulting in improving the tolerance of soybean seedlings to saline-alkali stress.

Photosynthetic Characteristics and Yield Components of Dwarf Sorghum Hybrid Liaoza 35

HUANG RuiDong, GAO Yue, ZHOU YuFei, WU Qi, ZHANG Jiao, SHANG PeiPei, ZHANG Zhuang, GAO MingYue, HAN Yi, XU WenJuan

Scientia Agricultura Sinica. 2017, 50(5): 822-829. doi:10.3864/j.issn.0578-1752.2017.05.005

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【Objective】Enhancing the mechanization level is now one of the most important goals in sorghum production. Important progress has been made in the dwarf sorghum hybrid breeding suitable for mechanization planting. The aim of this experiment is to investigate the photosynthetic characteristics and yield formation regularities of dwarf stalk sorghum hybrid, the results of the study will beneficial for the breeding and popularization of the new type sorghum hybrids. 【Method】 In this experiment, the newly bred dwarf sorghum hybrid Liaoza 35, suitable for mechanized production, was served as the main test material, with three planting densities (90 000 plants/hm², 120 000 plants/hm² and 150 000 plants/hm²), and the middle-high stalk sorghum hybrid Liaoza 19, the regional main cultivar with a similar growth duration, was used as the control (planting density was set as the suitable level of 105 000 plants/hm²), to explore the photosynthetic characteristics and yield formation regularities of the dwarf stalk sorghum hybrid through analysis of plant morphological, photosynthetic and ecological environment indexes and yield components of the sorghum population. 【Result】 At filling stage of the most flourishing population, with the increase of planting density, the dwarf sorghum Liaoza 35 showed significant reductions of light intensity, light transmittance, stomatal conductance and photosynthetic rate in the middle and lower parts of the canopy, and also significantly lower than those of the control hybrid Liaoza 19, but no significant difference in the above each index at the upper canopy between the two hybrid varieties. The electron transport rate (ETR), coefficient of photochemical quenching (qP) and the maximal photochemical efficiency (Fv/Fm) on the second leaf from the top of Liaoza 35 at flowering and filling stages showed a trend of decrease along with the increase of planting densities, while the initial fluorescence (Fo) showed a trend of increase, and all the fluorescence parameters of Liaoza 19 were better than those of Liaoza 35 on the condition of middle-high planting densities. Leaf area index increased with the increase of planting density, showing a trend of increase until anthesis and then gradual decline, with the fastest decline in the treatment of 150 000 plants/hm², however, a considerable leaf area index was also maintained at the maturity stage of Liaoza 35; leaf SPAD values declined significantly at lower parts of the canopy. Under high planting density conditions, the morphological, photosynthetic and ecological environment indexes of Liaoza 35 performed poorly compared to Liaoza 19, which might be associated with its plant type: dwarf in height, but no corresponding reduction of leaf number and no noticeable changing of leaf angle. With the increase of planting density, Liaoza 35 gave higher biological yield and grain yield, with significant reduction of grains per panicle but no significant change in 1000-kernel weight. Liaoza 35 produced the highest grain yield, 9 923.5 kg·hm^-2, at the planting density of 150 000 plants/hm², and over-yielded the control Liaoza 19. 【Conclusion】 Reasonable increase of planting density is a key to promote the grain yield increase of dwarf sorghum. However, high planting density may induce negative impacts on the photosynthetic characteristics and dry matter production in the population canopy, thus it is important to make the negative impact on the individual plants less than the positive effect on the population performance through improving sorghum plant type and the better management of water, fertilizers and planting densities to coordinate the relationship between the sorghum population and individual plants.

Timeliness Analysis of Crop Remote Sensing Classification One Crop A Year

LIU HuanJun, YU ShengNan, ZHANG XinLe, GUO Dong, YIN JiXian

Scientia Agricultura Sinica. 2017, 50(5): 830-839. doi:10.3864/j.issn.0578-1752.2017.05.006

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【Objective】Crop type remote sensing identification is a basis of crop cultivated area and crop growth analysis and yield estimation, and it is a very important driving force to promote the rapid development of modern agriculture. At the same time, it is also a basis for macro-regulation and control of understanding of agricultural conditions by the departments of agriculture as well as other related ones. At present, most of the present researches about agricultural remote sensing are limited to moderate or low resolution remote sensing images, which affect the accuracy of vegetable information extraction. The accuracy of vegetation information extraction can be improved by using high resolution multi temporal remote sensing images and selecting suitable classification methods. Clearly understanding of the timeliness and optimal classification method of crop remote sensing classification, acquire crop spatial distribution data quickly and accurately, and to provide a basis for crop quantitative remote sensing monitoring are the aims of the study.【Method】Based on the 20 remote sensing images covering the whole growth period of 5-10 months in Hulin, Heilongjiang province in 2014, the 16 m resolution NDVI time series curves were built by using 20 images. Different crops had different NDVI time series curves during the whole growth period. The decision tree classification model was established. After analysis of the images through serial threshold division, assisted with background data and expert knowledge, the areas and distributions of the land use and land cover information were extracted. Twenty images were used in order to classify the crops and the optimal phase was defined. Taking the farmland range as the rule, various classification methods for crop classification were compared. And it was also compared with the crop classification without extracting the farmland range by using several common methods of crop classification. Meanwhile, various classification methods including the maximum likelihood method, Mahalanobis distance method, neural network method, minimum distance method, support vector machine, spectral angle classification, and crop classification of principal component analysis were compared, and the data from the insured blocks were employed for the accuracy verification.【Result】 (1) In early July, the end of July to early August, and the end of September are the 3 key phases of crop remote sensing classification in the study area during the first quarter of the year. (2) The decision tree classification method had the highest accuracy in extracting land use cover information, the overall accuracy of classification was up to 94.01%, Kappa coefficient was 0.79. (3) In early June and early July, 2 images combined with classification of crops, the overall of classification accuracy was up to 90.24%, Kappa coefficient was 0.87. The combination of early June and early July images could be used to solve the timeliness of crop classification. (4) Combined with the image of Sep 21st, the overall accuracy was further improved, and the classification accuracy of soybean was improved obviously, so the maximum likelihood method was the best classification method, and the jointing stage was the best phase.【Conclusion】It was concluded that remote sensing images can be used to accurately classify crops in early July. Results of this study have expanded the application value of remote sensing data in the field of agriculture. It has guiding significance for one crop a year of the crop fast classification.

Subcellular Localization of the Ribosome-Inactivating Protein α-MC and Its Antiviral Effect on TMV

WEI ZhouLing, PENG HaoRan, PAN Qi, ZHANG YongZhi, PU YunDan, WU GenTu, QING Ling, SUN XianChao

Scientia Agricultura Sinica. 2017, 50(5): 840-848. doi:10.3864/j.issn.0578-1752.2017.05.007

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【Objective】The objective of this study is to obtain the alpha-momorcharin (α-MC) and pokeweed antiviral protein (PAP) by cloning, observe the cellular localization of which in Nicotiana benthamiana by heterologous expression, and to evaluate the effects of α-MC on inhibiting Tobacco mosaic virus (TMV) and the resistance defense response of N. benthamiana.【Method】Based on the published sequences of α-MC and PAP, primer pairs for cloning PAP and α-MC were designed. RT-PCR and gene cloning were used to obtain the target genes α-MC and PAP from the leaves of Momordica charantia and Phytolacca acinosa, respectively. First, subcellular localization of the α-MC and PAP was predicted through Wolf PSORT, then the fusion protein vectors for verifying the subcellular localization were constructed by fusing the α-MC and PAP to the N-terminal of the GFP and DsRed2, respectively. The α-MC was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the α-MC expressed leaves were inoculated with TMV-GFP. The accumulation of the virions and viral RNA were detected by indirect ELISA and real-time quantitative PCR (qRT-PCR). In order to understand the antiviral mechanism of the α-MC, the expression of plant defense-related genes including non-expressor of pathogenesis-related genes (NPR1, PR1, PR2) were evaluated by qRT-PCR. 【Result】 The length of genes α-MC and PAP obtained by RT-PCR were 861 and 939 bp, respectively. The subcellular localization showed that the coding proteins of α-MC and PAP were predicted by Wolf PSORT to distribute on the plasma membrane. Under the confocal laser scanning microscope, the fusion proteins α-MC-GFP and PAP-DsRed2 also distributed on the plasma membrane of the leaf epidermis cell of N. benthamiana, which is consistent with the predicted results. It was observed that the heterologously expressed PAP produced strong toxic effects on tobacco leaf cells, which led to necrosis of the cells, while the heterologously expressed α-MC showed no obvious toxic effect on tobacco leaf cells, which were kept intact. Additionally, following the heterologous expression of α-MC in N. benthamiana, TMV-GFP was inoculated. After 48 hours, there was no green fluorescence observed in the α-MC expressed leaves under UV light, but the green fluorescence could be observed in the control group. After 72 hours, sporadic green fluorescence was observed in the treatment group and the fluorescence in the control group started to spread. After 6 days, the green fluorescence spread to the spear leaf of the control group, while no obvious change was found in that of the treatment group. ELISA assay results showed that after 6 days of TMV-GFP inoculation, the value of OD₄₉₂ in the control group was over 10 times more than the value of samples under treatment. qRT-PCR data showed that the expression level of TMV in control group was 149 times than that of the level in group after treatment. Those data indicate that α-MC has a significant impact on both replication and movement of TMV. The expression levels of several defense-related genes in N. benthamiana leaves expressing α-MC with or without TMV were tested through qRT-PCR. Data showed that NPR1 was induced in both cases while the expression level was 2.5 times in plant with TMV injection than the one without injection. As to PR1 and PR2, the expression of these two genes was 5-7 times higher in plants with TMV injection. Combining all those data together, it was suggested that the resistance effect of heterologously expressed α-MC on plant viruses could induce the expression of responsive defense-related genes including NPR1, PR1 and PR2, resulting in much stronger plant defense response. 【Conclusion】 The heterologously expressed α-MC significantly inhibited TMV, activated the plant defense response, enhanced the defense response of N. benthamiana, and produced few toxicity to the plant cells. Therefore, the results will provide a reference for the development of new products for the control of plant viruses based on heterologous expression of α-MC.

Cloning, Expression and Enzymatic Characterization of Chitin Deacetylases from Hyphantria cunea

YAN XiaoPing, ZHAO Dan, GUO Wei, WANG Wei, ZHANG YaKun, GAO YuJie, ZHAO KunLi

Scientia Agricultura Sinica. 2017, 50(5): 849-858. doi:10.3864/j.issn.0578-1752.2017.05.008

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【Objective】 The objective of this paper is to study the function of the enzyme in the process of insect life, enzymatic characterization of chitin deacetylases from Hyphantria cunea and to provide a basis for understanding of its function in the insect life, and to provide new targets for the biological control strategy of H. cunea. 【Method】 The conserved domain of chitin deacetylase 2 sequences from three kinds of lepidoptera insects Bombyx mori, Helicoverpa armigera and Choristoneura fumiferana were analyzed and chitin deacetylase gene-specific primers were designed by homologous alignment. A cDNA clone was identified to contain cDNA sequence coding for chitin deacetylase by PCR amplification. The prokaryotic recombinant expression vector pET30a-HcCDA2 was constructed and cloned in E. coli, the protein expression was induced by IPTG and detected by SDS-PAGE electorphoresis. The recombinant baculovirus expression vector pFastBac-HcCDA1 and pFastBac-HcCDA2 were constructed, and liposome transfection method was used to transfect insect cells Hi5, then the P1, P2 and P3 virus were obtained, respectively. The P3 cells supernatant were collected and western blot analysis was applied to detect the chitin deacetylase 1 (from the previous study) and 2 protein expression. The recombinant proteins were crude purified with ammonium sulfate and then the enzymatic characterizations were studied with the 4’-nitroacetanilide (including the optimum temperature, the optimum pH and the effects of different metal ions on enzyme activity).【Result】 The full-length cDNA clone encoded chitin deacetylase 2 in H. cunea was identified (GenBank accession number: KT781841). The cDNA is 1.6 kb in length. The protein encoded by this cDNA is named HcCDA2 and the protein was shown a band on the SDS-PAGE gel with an apparent molecular weight of 61 kD. The specific antibodies reacting to HcCDA2 were obtained by immunizing rabbits. TheHcCDA1 and HcCDA2 were shown as 80 kD proteins in Hi5 cell line detected by Western blot analysis. The study of enzymatic properties showed that the two enzymes which were secreting expression in insect cells possessed catalytic activity, and the optimum temperature were both 50℃, when the temperature reached 80℃, the HcCDA2 almost lost its activity; HcCDA1 enzymatic reaction appropriate pH ranged from 7.0-9.0, and the optimum pH of HcCDA1 and HcCDA2 were both 8.0. Under the optimum reaction conditions, the enzyme activities of HcCDA2 protein were higher than HcCDA1 protein. Mg²⁺, Zn²⁺, Mn²⁺ and Ca²⁺ showed an inhibition on HcCDA1 and HcCDA2, an increasing Zn²⁺ concentration would lead to the enhancement of inhibitory effects on HcCDA1, but Mg²⁺showed a trend of increasing first and then decreasing on HcCDA1, as well as an increasing Mg²⁺ or Ca²⁺ concentration showed an inhibition on HcCDA2. Co²⁺ and Fe²⁺ showed activation on HcCDA1 and HcCDA2, Fe²⁺showed activation stably with increasing concentration, while Co²⁺ showed a trend of increasing first and then decreasing. 【Conclusion】 The gene HcCDA2 was cloned from H. cunea, expressed in E. coli shown as a 61 kD protein, and the HcCDA2 protein-specific polyclonal antibodies were obtained by immunizing rabbits. The active HcCDA1 and HcCDA2 proteins were obtained, and both of them showed a catalytic activity in vitro, the optimum temperature and pH were both 50℃ and 8.0, respectively.

Effect of Conservation Tillage Practices on Soil Microbial Spatial Distribution and Soil Physico-Chemical Properties of the Northwest Dryland

LI Tong, WANG ZiTing, LIU Lu, LIAO YunCheng, LIU Yang, HAN Juan

Scientia Agricultura Sinica. 2017, 50(5): 859-870. doi:10.3864/j.issn.0578-1752.2017.05.009

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【Objective】Conservation tillage has been applied widely in the arid region of Northwest China, it is an important technical measure of agricultural production, explorating the effect of conservation tillage on soil fertility and soil microbial community structure and will help the sustainable development of agricultural production. This experiment from the angle of the combination of soil physico-chemical properties and microorganism to investigate the effect of conservation tillage on soil microbial spatial structure, and the relevance of abundance of microbial communities and soil physico-chemical properties of dryland wheat field, providing a theoretical basis and practical support measures for the promotion of conservation tillage.【Method】In this experiment, the soil of arid region in Northwest China was used as the research object, compared to conservational plow tillage (PT), two kinds of conservation tillage methods, including chisel plow tillage(CPT) and zero tillage(ZT) were designed, the indicators of soil physico-chemical properties and the abundance of microbial communities were measured by using real-time PCR, and the relations between the spatial distribution of microbial communities and soil physico-chemical properties and conservation tillage were analyzed.【Result】Long-term conservation tillage had a significant effect on the environment of dryland wheat field, the abundance of soil fungi and bacterial communities had different effects with different tillage methods, they showed different levels of response to the three tillages. In different tillage methods, the spatial distribution of soil microbe was uneven, continuity was poor, higher spatial variability, and showed a strong spatial aggregated distribution. There were also significant effects of tillage method on soil physico-chemical properties and enzyme activities, compared with traditional tillage, chisel plow tillage and zero tillage could significantly improve soil clay, water, total nitrogen, ammonium nitrogen content and urease and invertase activities. The results of canonical principal component analysis (CPCA) showed that the changes of soil microbial community abundance and physico-chemical properties were mainly affected by tillage methods, and the soil microbial community abundance is closely related to physico-chemical properties, under the zero tillage mode, the content of soil clay, water and ammonium nitrogen significantly affected the distribution of soil bacterial community, and under chisel plow tillage mode, the content of soil soluble carbon and the activity of catalase significantly affected the distribution of soil fungal communities.【Conclusion】Using conservation tillage in dryland wheat field could affect the abundance and the spatial distribution of soil microbial communities, and significantly affected soil physico-chemical properties, and then affecting the spatial structure of soil microbes. Simultaneously, soil moisture and the contents of carbon and nitrogen were respectively significantly affected soil bacteria and fungi community abundance.

Leaf Water Potential Estimating Models of Winter Wheat Based on Hyperspectral Remote Sensing

CHEN ZhiFang, SONG Ni, WANG JingLei, SUN JingSheng

Scientia Agricultura Sinica. 2017, 50(5): 871-880. doi:10.3864/j.issn.0578-1752.2017.05.010

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【Objective】A model for fast, non-destructive and accurately monitoring leaf water potential of winter wheat was established with hyperspectra technology, it will provide a scientific basis for the precision irrigation management of winter wheat.【Method】Using the field trials of different water treatments, the canopy spectral reflectance, leaf water potential and soil moisture were synchronously determined in the growth period of winter wheat. Then the correlation between the hyperspectral vegetation indices and leaf water potential was analyzed. Using correlation analysis, regression analysis and other methods, four inversion models were constructed for estimating leaf water potential based on different water treatments.【Result】The canopy spectral reflectance of winter wheat had significant change characteristics in different water treatments and growth periods. In the visible wave band, the canopy reflectance of winter wheat was reduced gradually along with the increase of soil water content. But, in the near-infrared wave band, the canopy reflectance was increased with the increase of soil water content. With the development of wheat growth period, in the near-infrared wave band, the canopy reflectance at heading stage was higher than the reflectance at jointing stage. And after the filling stage, the reflectance of red and blue band was rose faster. The correlation between four vegetation indices and leaf water potential was all reached the significant level (P<0.05), and its absolute values of correlation coefficient were all above 0.711. Four vegetation indices could be used for quantitative monitoring leaf water potential of winter wheat. Under the field capacity of 70%, the absolute values of correlation coefficient |r| between the vegetation indexes of OSAVI and EVI and the leaf water potential were 0.75 and 0.771, respectively, they were lower than the |r| between the vegetation indexes of NDVI and RVI and leaf water potential, which the values of |r| were 0.808 and 0.896, respectively. But, under the field capacity of 50%, the results were just the opposite. The |r| between the vegetation indexes of OSAVI and EVI2 and the leaf water potential were 0.857 and 0.853, respectively, which were higher than the |r| between the vegetation indexes of NDVI and RVI and the leaf water potential, which the values of |r| were 0.711 and 0.792, respectively. The estimation values of 45 samples in prediction set were close to the measured values, the range of R², MRE, and RMSE were 0.616-0.616, -17.50%—-12.52% and 0.102-0.133, respectively. Under the 70% FC water treatment, the estimating model of leaf water potential based on EVI2 had the highest R², the value of R² was 0.922, and under the 60% FC and 50% FC water treatments, because of considering the influence of soil background, the inversion models of leaf water potential based on OSAVI had the highest R², the values of R2 were 0.922 and 0.856, respectively.【Conclusion】All the four vegetation indices could be used for quantitative monitoring leaf water potential of winter wheat. But, when the leaf water potential estimating models were built for different water treatments, the influence of soil background on canopy spectral should be considered. The research results could provide a technical basis for wheat precision irrigation management and also provide supporting models for the parametric inversion of the onboard data.

Effect of Hydrogen-Rich Water Soaked Cucumber Seeds on Cold Tolerance and Its Physiological Mechanism in Cucumber Seedlings

LIU FengJiao, CAI BingBing, SUN ShengNan, BI HuanGai,AI XiZhen

Scientia Agricultura Sinica. 2017, 50(5): 881-889. doi:10.3864/j.issn.0578-1752.2017.05.011

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【Objective】Hydrogen (H₂), a newly discovered gas signal molecules, is involved in plant stress responses to high temperature, drought, salt damage, heavy metals and other kinds of adversity. The purposes of this study are to elucidate the regulatory mechanism of hydrogen-rich water (HRW, H₂ donor) on chilling tolerance in cucumber seedlings and provide technical guidance to improve the adaptation of cucumber to low temperature in solar-greenhouse. 【Method】 ‘Jinyou 35’ cucumber seedlings were used as experimental materials. Seeds were soaked with HRW or distilled water (control), respectively, for 8 h, and germinated on moist filter paper in the dark at 26℃ for 2 days, then grown in vermiculite in a solar-greenhouse for 15 days (day/night air temperature was 24-30℃/18-24℃, and RH 75%-90%). At 2-leaf stage, the HRW and the control seedlings were exposed to low temperature (day/night temperature was 8℃/5℃). Young fully expanded leaves were sampled for analysis on 0 d, 1 d, 3 d, and 5 d after transferring from control to stress condition. 【Result】The results showed that chilling stress significantly increased the electrolyte leakage (EL), chilling injury index, contents of hydrogen peroxide (H₂O₂) and malondaldehyde (MDA), and superoxide anion (O₂^-) production rate. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as the contents of glutathione (GSH), ascorbic acid (AsA), proline and soluble sugar increased in the early days of chilling stress, but subsequently decreased. The relative water content trended to decrease in the chilling days. The increase in EL and chilling injury index were lower in HRW treated seedlings than in the control seedlings. Simultaneously, the HRW treatment showed a decrease in H₂O₂and MDA contents, and O₂^- production rate, while revealed an increase in the activities of SOD, POD, CAT, APX and GR, as well as the GSH and AsA contents, compared with the control. At the end of stress (5 d), the EL and chilling injury index of HRW treatment declined by 11.3 percentage points and 15.9%, respectively, than those of the control. The H₂O₂and MDA contents and O₂^- production rate of the HRW-treated seedlings were 29.4%, 9.9% and 54.3% lower than those of the control, respectively. However, the activities of SOD, POD, CAT, APX and GR of HRW treated seedlings were 12.6%, 20.1%, 20.9%, 53.0%, and 58.1% higher, and the GSH and AsA contents enhanced by 24.0% and 17.6%, respectively, than those of the control seedlings. Compared with the control, the HRW treated seedlings showed lower extent of decrease in the relative water content, and revealed higher contents of proline and soluble sugar. After 5 d of chilling stress, the HRW treatment increased by 6.4 percentage points, and the proline and soluble sugar contents were 23% and 41.5% higher, respectively, than those of the control. 【Conclusion】 Soaking seeds with HRW can improve the cold resistance in cucumber seedlings, and the main mechanisms were: (1) HRW enhances the antioxidant system activity and reduces the reactive oxygen species (ROS) accumulation under chilling stress, and consequently alleviates the injury of membrane lipid peroxidation in cucumber seedling; (2) HRW slows the dehydration rate through improving the osmotic adjustment ability, and thereby remains the normal physiological function for a long time in cucumber seedlings under chilling stress.

Composition and Concentration of Flavan-3-ols in Berry Peel of 11 Chinese Wild Grape Species

YAN Jing, JIANG Yu, FAN XiuCai, JIANG JianFu, ZHANG Ying, SUN HaiSheng, LIU ChongHuai

Scientia Agricultura Sinica. 2017, 50(5): 890-902. doi:10.3864/j.issn.0578-1752.2017.05.012

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【Objective】China is one of the countries rich in resources of Vitis germplasm in the world, most of their berries are little in size, with thick dark-red peels, thus they are expected to be utilized for high quality wine production. While as a critical component to wine quality, flavan-3-ols in peel of Chinese wild grapes seldomly has been researched. In the present paper, the difference of the composition and concentration of flavan-3-ols in berry peel of different Chinese wild grape species was evaluated, with the aim of leading to better choice of raw materials for wine making and evaluation of Chinese wild grape germplasm.【Method】 Ninety-eight cultivars or clones of eleven Chinese wild species were selected, including Vitis piasezkii, V. davidii, V. pseudoreticulata, V. betulifolia, V. heyneana, V. romanetii, V. heyneana subsp. ficifolia, V. amurensis, V. adenoclada, V. amurensis var. yanshanensis and V. adsticta. Vitis. vinifera L.‘Cabernet Sauvignon’ was chosen as control. Mature berries were collected from the National Grape Germplasm Repository in Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences in 2015. The composition and concentration of flavan-3-ols in berry peel of selected samples were determined by reversed-phase UPLC after acetone extraction and acid-catalysis in the presence of excessive phloroglucinol. 【Result】 The results are as follows: The contents of flavan-3-ols from different species were different. Among the eleven species, the contents of flavar-3-ols in V. heyneana subsp. ficifolia were much higher in berry peel than the control and the other detected species, while that in V. betulifolia and V. piasezkii were the lowest in berry peel, only 64% of the control. The compositions of falvan-3-ols in berry peel of Chinese wild grapes was similar to that of the control, and composed by catechin (C), epicatechin (EC), epigallocatechin (EGC) and epicatechin-3-O-gallate (ECG). In berry peel, EGC was the major constituent representing around 60% of total flavan-3-ols, with EC accounting for around 35% of total flvan-3-ols, while C and ECG represented less than 5%. These four constitutional units can not only being as terminal subunits but also as extension subunits of polymeric flavan-3-ols. The flavan-3-ols in berry peel of Chinese wild species was mainly existed as polymers, and representing above 90% of total flavan-3-ols. The mean degree of polymerization (mDP) of different species was different, among the detected species, V. amurensis had the highest level of mDP above 50, while V. betulifolia was only 25 and the other species were between 25 and 45. Of these flavan-3-ol compounds, EGC is synthesized from the F3′5′H-mediated branch pathway and so is grouped into the 3′,4′,5′-substituted flavan-3-ols, whereas C, EC and ECG are from the F3′H-mediated branch and are grouped into 3′,4′-substitued flavan-3-ols. Compared with the other species, V. heyneana and V. romanetii had a much lower percentage of total 3′,4′-substituted flavan-3-ols (C, EC and ECG) and a significantly higher percentage of 3′,4′,5′-substituted flavan-3-ols (EGC). Principle component analysis revealed that there was a certain distinction among the wild species, while V. davidii was notably from the other species.【Conclusion】 Compared with the V. vinifera L.‘Cabernet Sauvignon’, some special cultivars or clones of the V. davidii, V. betulifolia, V. heyneana subsp. ficifolia, V. amurensis and V. adenoclada possess much higher flavan-3-ols content in berry peel, which hold a great potential be used for breeding new materials with high level of flavan-3-ols. Besides, the content of flavan-3-ols varied among different species, which would act as an additional index to evaluate the quality of Chinese wild grape germplasm.

Effect of Extraction Time on Characteristics of Gelatin and Secondary Structure of Chicken Bone Protein

YUE JianYing, WANG JinZhi, ZHANG ChunHui, DU GuiHong, XU Xiong

Scientia Agricultura Sinica. 2017, 50(5): 903-912. doi:10.3864/j.issn.0578-1752.2017.05.013

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【Objective】Hot-pressure extraction was utilized to prepare chicken bone protein (CBP) to study the effect of extraction time on gelatin properties. The study was expected to provide a reference for the preparation technology of edible gelatin by using chicken bone by-products. 【Method】After being adjusted to the same concentration of protein, CBP at different extraction times were incubated at 4℃ to form gelatin. The content of total soluble substance (TSS), crude protein and hydroxyproline (Hyp), secondary structure of protein and distribution of molecular weight (MW), as well as color of gelatin and gel strength were studied. Furthermore, correlation analysis between indexes of CBP was also performed to establish possible linkages between physicochemical properties and gelatin properties of CBP.【Result】The results showed that hot-pressure extraction time had a significant effect on the yield of CBP (P＜0.05), leading to the variable structure of protein and consequently affected the properties of gelatin made by CBP. The contents of TSS, crude protein and Hyp were increased significantly during extraction (0-120 min) (P＜0.05). The result of MW distribution suggested that protein experienced dramatic degradation from 40 to 120 min (P＜0.05). The proportion of MW between 10 and 30 kDa decreased from 59.82% (40 min) to 13.99% (120 min) while the ratio of MW ＜10 kDa increased from 35.46% (40 min) to 86.01% (120 min). In addition, the extraction time caused the denaturation of protein and thus leading to the change of secondary structure. The CBP was composed of 100 percent of α-helix (0-40 min), and then β-sheet emerged at 60 and 90 min (9.9% and 17.6%, respectively) but disappeared at 120 min, which indicated that the secondary structure of protein experienced transformation during extraction. Results of CBP gelatin properties suggested that CBP at 0 min could not form a gelatin and thus had no gelatin properties. Gel strength at 20, 40 and 60 min was at same level and better than CBP gelatins of 90 and 120 min. In addition, transparency of CBE solutions was significantly impacted by extraction time, which increased sharply during hot-pressure extraction (20-120 min) (P＜0.05). Meanwhile, extraction time significantly affected the color of gelatins (P＜0.05) and gelatin at 40 and 60 min met the requirements of high lightness, low yellowness and high whiteness. However, water holding capacity was insensitive to the extraction time (P＞0.05). Correlation analysis showed that the content of Hyp, degradation of protein were considerably related to gelatin properties (P＜0.05).【Conclusion】In conclusion, extraction time could significantly affect the gelatin properties prepared by CBP at different extraction times. Given yield of protein, color of gelatin, gel strength, etc., CBP extracted at 40 min and 60 min were better proper for preparation of gelatin.

Metagenomic Analysis of Bacterial Phases of Catfish Fillets Under Different Storage Conditions

ZHU YingChun, WANG Yang, FAN XiaoPan, MA LiZhen, WANG KaiLi

Scientia Agricultura Sinica. 2017, 50(5): 913-923. doi:10.3864/j.issn.0578-1752.2017.05.014

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【Objective】The objective of this experiment is to analyze the dynamic changes of bacterial phase of catfish fillet stored under different storage conditions, evaluate the effect of natural preservative on the special spoilage bacteria. 【Method】There were 8 groups in this experiment including fresh catfish (Calarias gariepinus) fillets (CK0), AP (air-package) catfish fillets stored at (4±1)℃ for 4 and 7 d (AP4, AP7), MAP (modified atmosphere package, 60%CO₂/40%N₂) catfish fillets stored at controlled freezing-point temperature (0.7±0.02)℃ for 10 and 30 d(MAP10,MAP30), MAP catfish fillets added with 5% natural preservative stored at controlled freezing-point temperature (0.7±0.02)℃ for 10, 30 and 40 d (MAPP10, MAPP30 and MAPP40). The natural preservative consists of 0.5% chitosan, 0.1% propolis, 0.075% lysozyme, 0.075% Nisin and 0.5% tea polyphenol. Microbial16S rDNA of different samples were sequenced in metagenomics analysis, bacterial composition and abundance of these groups were compared. Alpha diversity and principal component analysis were carried out to investigate the effect of the natural preservative, package and storage temperature on bacterial phase changing.【Result】Totally 25 phylum, 433 genuses of bacteria were identified in 8 sample groups. The same as CK0, the principal genus in MAP10, MAPP10, MAPP30 was Actinomyces, while it was Aeromonas (70.49%) and Pseudomonas (59.01%) in AP4 and AP7, respectively. From the respect of microbiology, MAP + controlled freezing-point temperature was better than AP + low temperature storage. During the storage, the abundance of Lactococcus increased sharply from 15.78% (MAP10) to 82.85% (MAP30), while in the MAPP group (MAPP10 and MAPP30) which supplemented with natural preservatives Lactococcus rarely could be detected. 【Conclusion】MAP + controlled freezing-point storage significantly lowered the bacterial abundance and diversity, therefore is useful to prolong the shelf life of catfish fillets. The components of tea polyphenol, Nisin and chitosan in the natural preservative used in this study markedly inhibited the growth and the multiplication of Lactococcus which is the special spoilage bacteria in the MAP fillets stored at controlled freezing point. This is important for improving the security of catfish meat against microbial contamination through precise target inhibition.

Effect of Heat Stress on the Expression of Cyt-C and Caspase-3 in Boar Testis

FAN XiaoRui, ZHANG Zhen, XI HuaMing, LIANG YaJun, HE JunPing

Scientia Agricultura Sinica. 2017, 50(5): 924-931. doi:10.3864/j.issn.0578-1752.2017.05.015

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【Objective】 Elevated ambient temperature has detrimental effects on sperm quality in mammalian species including pig. But the molecular mechanism has not yet well understood. The aims of present study were to investigate the effect of heat stress on the expression of Cyt-C and Caspase-3 in boar testis. 【Method】 Nine mature boars (Landrace, 18 months of age) were used in the study. Three boars were subjected to 37-40℃ for 3 h daily for 7 days (heat stress 7 d, HS7d). Three boars were subjected to 37-40℃ for 3 h daily for 42 days (heat stress 42 d, HS42d). Following daily heat treatment the boars were driven back to normal house (20-27℃). The other 3 boars were kept in the normal house as control group (20-27℃). All boars were castrated and the testes were harvested. Western blot, QRT-PCR, and immunohistochemistry were used to analyze the expression of Cyt-C and Caspase-3. 【Result】 QRT-PCR showed that mRNA expression of Cyt-C and Caspase-3 were all increased in HS7d group and HS42d group compared with the control. And the relative expression of Cyt-C and Caspase-3mRNA in HS7d group was the highest. Western blot showed that the protein expression of Cyt-C and Caspase-3 was all increased in HS7d and HS42d as well, compared with the control group. And the protein expression of Cyt-C and Caspase-3 in HS7d group was the highest. Immunohistochemistry found that Cyt-C localized in the cytoplasm of Leydig cells, Sertoli cells and all stages of germ cells. In spermatogonia, the Cyt-C expression was low, while in postmeiosis germ cells, the expression of Cyt-C was higher. Heat treatment resulted in a more diffuse cytoplasmic localization of Cyt-C, which suggested that Cyt-C was released from mitochondria to the cytoplasm. In Leydig cells and spermatogonia, the Caspase-3 expression was low. A large number of postmeiosis germ cells showed Caspase-3 positive immunoreactive staining. Heat stress resulted in some Sertoli cells Caspase-3 positive staining and Caspase-3 positive with nucleus staining increased.【Conclusion】Elevated ambient temperature induced the increase of the expression of Cyt-C and Caspase-3, and the changes of cellular location, which indicated that the expression change of Cyt-C and Caspase-3 may be associated with the sperm quality decrease induced by heat stress.

Screening of Optimal DNA Extraction Methods and the Bacterial Community Composition of Yak Rumen Revealed by High 16S rRNA Throughput Sequencing

YANG QiYue, HUANG Yong, CHEN YaBing, LIU LuoChuan, LI Jian, LAN DaoLiang

Scientia Agricultura Sinica. 2017, 50(5): 932-941. doi:10.3864/j.issn.0578-1752.2017.05.016

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【Objective】The objective of this study is to determine the optimal DNA extraction methods for yak rumen bacterial genome and preliminarily identify the bacterial community of yak rumen.【Method】Physical method (bead-beating, freeze-thaw), chemical method(CTAB, SDS) and enzymatic method (lysozyme, protease K) were combined with the characteristics of rumen bacteria, 9 different methods (method 1, CTAB-SDS-Lysozyme; method 2, CTAB-SDS-Lysozyme-freeze-thaw; method 3, CTAB-SDS-Lysozyme-bead-beating; method 4, CTAB-Lysozyme; method 5, CTAB-Lysozyme-freeze-thaw; method 6, CTAB- Lysozyme-bead-beating; method 7, SDS-lysozyme; method 8, SDS-lysozyme- freeze-thaw; method 9, SDS-lysozyme-bead-beating) were generated to extract the rumen bacterial DNA, besides, method 10, QIA amp DNA Stool Mini Kit, was also used as reference. The above 9 methods were compared and analyzed based on DNA concentration, purity, DNA electrophoretogram and 16S rRNA high throughput sequencing results, and the bacterial community of yak rumen was preliminarily identified based on the sequencing results.【Result】The comparison results of different DNA extraction methods efficiency showed that the combined beat-beating and freeze-thaw could significantly increase cell lysis efficiency and DNA yield under the same chemical and enzymatic conditions. Methods 3 and 6 obtained high DNA concentration and purity, methods 7, 8, 9, and 10 had lower volume of DNA than other methods because of the lack of CTAB. In addition to methods 2 and 8 as their PCR products were too weak or not detected, the PCR products of rest methods all had suitable objective strap size and concentration which met the requirements of 16S rRNA high throughput sequencing. After high throughput sequencing, a total of 191 349 raw reads were obtained, and 171 231 clean reads were obtained after quality control. Rare faction curves analysis showed that the amount of reads reached the saturation level, which could completely reflect the bacterial community species of the samples. OTU-based taxology and the diversity index analysis showed that methods 6 and 10 contained more abundant bacteria. The comparison of the effect of different extraction methods on Gram-positive bacteria showed that method 6 presented a comparatively higher capability in lysing cell wall of Gram-positive bacteria. Above all, the DNA yield, OTUs number, bacteria diversity, and excellent cell-breaking capability of method 6 (CTAB-Lysozyme- bead-beating) was better than that with other methods. The bacterial community structure analysis showed that the number of taxa in yak rumen contained approximately 21 phyla, 35 classes, 75 families, 112 genera. The bacteria community of higher abundance included Bacteroidtes (64%), Firmicutes (20%), Spirochaetae (2.3%), Proteobacteri (1.8%) and Fibrobacter (1.7%). Comparative analysis showed that there are some variation in the bacterial community between yaks and cattle, which may be attributed to different diets and habitats.【Conclusion】This study screened out an optimal DNA extraction method (method 6,CTAB-Lysozyme-Bead-beating) for yak rumen bacterial genome using 16S rRNA high-throughput sequencing, and the bacterial community structure of yak rumen was preliminarily evaluated. Results of the study provide a foundation for identifying the specificity of rumen microbial community and discovering distinct gene resource in yak.

Designing and Validation of the Remote Monitoring System for Pigs’ Survival Based on IoT Technology

CHEN GuiPeng, QIN WenJing, DING Jian, WAN MingChun, GUO LeiFeng, WANG WenSheng

Scientia Agricultura Sinica. 2017, 50(5): 942-950. doi:10.3864/j.issn.0578-1752.2017.05.017

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