Table of Content

16 January 2018, Volume 51 Issue 2

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Differential Expression of Grain Pigment Related Genes of Guizimai No.1

XU Xi, REN MingJian, LI LuHua, YANG XiCui, XU RuHong

Scientia Agricultura Sinica. 2018, 51(2):  203-216.  doi:10.3864/j.issn.0578-1752.2018.02.001

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【Objective】 The objective of this study is to investigate the transcriptome differences after and before purple- changing periods of grain at filling stage of Guizimai No.1, explore the key genes and enzymes that contribute to the biogenesis of anthocyanin, and then enrich the transcriptome data of grain pigment in wheat, provide references for the cloning and expression of the transcription factor. 【Method】 RNA-seq, construction library and quality assessment were carried out for two periods before and after purple-changing of Guizimai No.1 by using the Illumina Hiseq 2000^TM sequencing platform, and the sequencing results was analyzed by bioinformatics. TTM was used to standardize the read count data, then DEGseq was used to analyze the difference, and the q-value＜0.005 and | log₂ (fold change) |＞1 were set as the threshold. The differential expression genes (DEGs) were obtained through selecting, in accordance with the transcriptome sequencing, then these DEGs were analyzed by BLAST search, NR annotated, GO functional enrichment and KEGG pathway method to find out the key genes and enzymes associated with anthocyanins, and combined qRT-PCR to verify the expression level of the key genes and key enzymes in different periods, finally the information of these key genes was mastered. 【Result】 The RNA-seq results showed that 13.36 G and 12.69 G clean bases were obtained, 106 906 108 and 101 547 534 clean reads accounted for 93.73% and 94.90% of the raw reads after and before purple-changing of Guizimai No.1, respectively. Clean reads were spliced by Trinity, totally 170 396 transcripts were obtained with a length of 119 020 625. There were 119 572 Unigenes after splicing clean reads. In the BLAST search, 86 004 (71.92%) Unigenes out of 119 572 high quality unique sequences had at least one significant match to existing gene models. According to Unigenes’ Nr database alignment, at least 5 Unigenes with similar gene identities and known sequence homologies to Aegilops tauschii, Triticum urartu, Brachypodium distachyon, Hordeum vulgare, Triticum aestivum, and so on were identified. The results of KOG database alignment showed that the annotated genes were classified according to 26 groups in KOG, and the greater percentage of generally functional genes, posttranslational modification and transport, molecular chaperones and translation, ribosomal structure and biosynthesis was 15.79%, 14.51% and 10.54%, respectively. A total of 643 DEGs were found, 236 DEGs were up-regulated and 407 DEGs were down-regulated. GO commentary indicated that there were 44 terms in accordance with biological process, cellular component, molecular function of the next level of classification, the differential genes significantly enriched in the carbohydrate metabolism process (GO: 0005975, 16.03%), stress response (GO: 0006950, 10.83%) and hydrolase activity (GO: 0016787, 34.84%) and other categories. KEGG pathway enrichment analysis showed that the 353 different genes were enriched in 153 related pathways, among them, the pathways of starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis were significantly enriched. There were 66 genes related to flavonoid biosynthesis, and two up-regulated Unigenes, involving two key enzyme genes of CHS, ANS. log₂ (fold change) were 3.4164 and 2.1258, respectively. The qRT-PCR results showed that the expression of CHS and ANS after purple-changing was significantly up-regulated, which was consistent with the results of RNA-Seq analysis, RNA-seq results were reliable. 【Conclusion】Compared the RNA-seq after and before purple-changing periods of Guizimai No.1 grain, a large number of Unigenes and DEGs were obtained. It is identified that the two key enzyme genes (CHS and ANS) in flavonoid metabolism pathway play a significant role in the regulation of anthocyanin synthesis in Guizimai No 1.

Over-Expression of Yeast PAC1 Confers Enhanced Resistance to Soybean mosaic virus in Transgenic Soybean

NIU Lu, ZHAO QianQian, YANG Jing, XING GuoJie, ZHANG Wei, HE HongLi, YANG XiangDong

Scientia Agricultura Sinica. 2018, 51(2):  217-225.  doi:10.3864/j.issn.0578-1752.2018.02.002

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  【Objective】Soybean mosaic virus (SMV) is one of the most prevalent viral diseases in major soybean growing areas of China and cause significant yield losses and quality deterioration of soybean seeds. Ribonuclease PAC1 was known to bind and degrade the dsRNA molecules generated during plant RNA viruses or viroids replication, thus effectively inhibited replication and accumulation of viruses or viroids in host plants. This feature of PAC1 provides an effective target gene for the creation and cultivation of the broad-spectrum anti RNA virus and viroid transgenic crops. The objective of this study is to introduce a PAC1 derived from Schizosaccharomyces pombe into soybean by transgenic technique, research the effects of over-expression of PAC1 onSMV resistance of soybean, and to provide a basis for the development of SMV-resistant soybean cultivars. 【Method】 The PAC1 was subcloned into the binary expression vector pCAMBIA3300 by restriction enzyme digestion and ligation to generate recombinant construct pCAMBIA3300-PAC1. In the resulting construct, the PAC1 was located between the constitutive promoter CaMV 35S and the terminator NOS. The construct also contained a BAR which confers resistance to the herbicide phosphinothricin (PPT). The soybean cultivar Williams82 was used for Agrobacterium-mediated transformation. Based on the detection of LibertyLink^® strip, PCR and herbicide spray (500 mg·L^-1 Basta), the integration and copy numbers of the exogenous gene in transgenic lines were further analyzed by Southern blot hybridization. Mechanical rub inoculation was performed to evaluate the resistance of the T₂ and T₃ transgenic lines and agronomic traits were analyzed under field conditions. Furthermore, the accumulation of SMV in the transgenic lines 28 days after SMV inoculation was analyzed using qRT-PCR. 【Result】A total of 76 PPT-tolerant plants were generated from  2 600 explants after Agrobacterium-mediated transformation. Among them, 65 regenerated plants were confirmed positive as shown by PCR and LibertyLink^® strip analysis, and the transformation efficiency was 2.48%. The transgenic plants in subsequent generations (T₁ to T₃) were further screened by spraying 500 mg·L^-1 Basta. The transgenic plants showed no visible phenotype change 7 days after treatment, whereas the non-transgenic (NT) plants exhibited symptoms such as chlorosis or necrosis. Southern blot analysis showed that the exogenous gene was integrated into soybean genome with 1-2 copies of T-DNA insertions in the selected transgenic plants. Resistance evaluation by rub inoculation with SMV showed that the NT plants displayed serious mosaic patterns and curling leaves, while only mild mosaic symptom was observed on some of the leaves of the transgenic plants (35 days after inoculation). Moreover, the transgenic lines had significantly lower average disease indices (11.11-22.22) than the non-transformed (NT) control plants (36.81-46.24) and showed enhanced and stable resistance to SMV. The qRT-PCR result further confirmed that the SMV CP expression in the transgenic plants was significantly decreased than that of the NT control 28 days after SMV inoculation. The investigation of agronomic traits showed that there was no significant difference in the leaf shape, flower color, seed color, hilum color, plant height, node number, podding height, maturity period and weight per 100 seeds between the transgenic lines and NT control plants without inoculation SMV. 【Conclusion】The over-expression of PAC1 significantly inhibited the accumulation of SMV and the development of the symptoms, thus enhancing the resistance level to SMV in transgenic soybean plants.

Association Mapping of Fall Dormancy with SSR Markers in Alfalfa (Medicago sativa L.)

LIU XiQiang, ZHANG Han, WANG XueMin, YI DengXia, WANG Zan

Scientia Agricultura Sinica. 2018, 51(2):  226-232.  doi:10.3864/j.issn.0578-1752.2018.02.003

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【Objective】The aim of this study was to identify simple sequence repeat (SSR) loci associated with fall dormancy in alfalfa (Medicago sativa L.) for understanding its inheritance pattern and molecular mechanism.【Method】The association population was composed of a total of 321 tetraploid genotypes from 75 alfalfa accessions, and each of the Chinese accessions was represented by six to eight genotypes, while the remaining ones included three to four genotypes. All the 321 materials were genotyped using 85 SSR markers randomly distributed on alfalfa genome. The phenotyping data of fall dormancy trait of these alfalfa accessions was obtained in 2014 and 2015, and used together with the SSR genotyping results for an association mapping by using General Line Model (GLM) and Mixed Line Model (MLM) models.【Result】It was showed that the fall dormancy trait in alfalfa was significantly influenced by genotypes, years, and genotype × year interactions. The fall plant heights ranged between 5.1 cm and 55.1 cm, and between 3.5 cm and 44.9 cm, averages of 22.4 cm and 15.2 cm, and coefficients of variation at 45.5% and 43.7% in 2014 and 2015, respectively. This trait showed a normal or nearly normal distribution in both 2014 and 2015 and had a relatively high broad-sense heritability at 0.71. The MLM model which adequately controlled false positives identified a total of 12 and 11 significant associations accounting for 2.42% to 6.73% and 2.45% to 4.81% of the phenotypic variances in 2014 and 2015, respectively. Among them, four loci of m83_157 on Chr. 2, m525_230 and m525_231 on Chr. 3, and m429_245 on Chr. 4 were detected in the two years by using the two models.【Conclusion】It was concluded that the four association loci related to fall dormancy in alfalfa were identified, and would be subjected to functional verification so that they could be eventually used for alfalfa marker assisted selection breeding.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Stem Flow Influencing Factors Sensitivity Analysis and Stem Flow Model Applicability in Filling Stage of Alternate Furrow Irrigated Maize

DU Bin, HU XiaoTao, WANG WenE, MA LiHua, ZHOU ShiWei

Scientia Agricultura Sinica. 2018, 51(2):  233-245.  doi:10.3864/j.issn.0578-1752.2018.02.004

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【Objective】The artificial neural network is used to predict the stem flow of maize with different treatments, which provides a new idea for estimating the water consumption of maize so as to make reasonable irrigation plan. 【Method】 The summer maize, variety of Xinong 985, was selected for study. The test set three treatments: alternative furrow irrigation high water treatment (alternate furrow irrigation, AFI1), alternate furrow irrigation low water treatment (alternate furrow irrigation, AFI2), and conventional furrow irrigation (conventional furrow irrigation, CFI). AFI1 and AFI2 each irrigation amount was 2/3 and 1/2 irrigation amount of CFI, respectively. In this paper, an artificial neural network is established to estimate the corn stem flow rate, and the model is compared with the principal component regression model, and the accuracy and stability of the two models are evaluated.【Result】(1) The response of different treatments to environmental factors was different. Meteorological conditions were the main factors affecting stem flow of the AFI1 treatment, and soil moisture was main factor affecting stem flow of the AFI2 treatment. (2) The influence of water content in different soil layers on stem flow was also different. It was found that the moisture contents of 10-20 cm and 20-30 cm layers were most correlated with maize stem flow. By using the uncertainty analysis method further analysis: For CFI and AFI1 treatments, 20-30 cm layer soil content was most closely related to sap flow changes, then 10-20 cm layer soil content. And for AFI2 treatment, 10-20 cm layer soil content was most closely related to sap flow changes, then 20-30 cm layer soil content; (3) After comparing the R² , RMSE and d-factor values of two models, the ANN model was more stable and accuracy, which was found to be the best model to predict the stem flow.【Conclusion】Compared with traditional methods, artificial neural network model can greatly enhance the prediction accuracy of stem flow, and it can provide some guidance for making reasonable irrigation plan of maize.

Temporal and Spatial Changes in Yield and Quality with Grain Sorghum Variety Improvement in China

LI SongBo, TANG ChaoChen, CHEN Feng, XIE GuangHui

Scientia Agricultura Sinica. 2018, 51(2):  246-256.  doi:10.3864/j.issn.0578-1752.2018.02.005

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【Objective】 In order to provide a theoretical basis for variety breeding and production, yield and quality relevant traits of accredited grain sorghum varieties were investigated from 1977 to 2016. 【Method】 The information of a total of 324 grain sorghum varieties authorized by state and province were obtained through yearbook and related articles search, and their yield and quality trials in regional trial were analyzed. 【Result】 In temporal evolution, sorghum plant height decreased annually by 1.36 cm whereas yield of regional trial increased annually by 69.1 kg·hm^-2 in 40 years. The content of starch and tannin were obviously increased (P＜0.05), whereas protein content decreased markedly (P＜0.05). Lys and fat content showed no significant trend over time. In spatial distribution, average growth period of grain sorghum was found a large difference in different zones. Higher spike grain weight (105.4 g) in the spring and summer sowing zone, contrary to south zone (64.6 g). 1 000 grain weight exhibited significant difference among in different zones (P＜0.05), and visualized maximum value of 30.3 g in spring-seeding and late-maturing zone, and expressed minimum value of 22.6 g in south zone. Average starch content was 74.2%, while fat content was 3.5% in spring-seeding and early-maturing zone, as well as spring-seeding and late-maturing zone. Tannin content was assumed the highest in south zone. Protein and Lys content showed no significant difference in different zones. 【Conclusion】 More significant measures should be taken for 1000 grain weight improvement greatly based on dwarfing of stalk and breeding special varieties. It is essential for liquor-making to develop with appropriate high starch content, reasonable content of protein and Lys, and especially in improving tannin content. The major approach of forage sorghum development should be the decreasing of tannin content and increasing of protein and Lys content based on development of high starch content.

PLANT PROTECTION

The Function of the Carbon Metabolism Regulator FgCreA in Fusarium graminearum

HOU Rui, WANG ChenFang

Scientia Agricultura Sinica. 2018, 51(2):  257-267.  doi:10.3864/j.issn.0578-1752.2018.02.006

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【Objective】The objective of this study is to knock out the FgCreA of Fusarium graminearum, which is the carbon source metabolic regulation factor, to research the vegetative growth, sexual reproduction and pathogenicity of the knock out mutant, and to provide a basis for carbon source metabolic mechanism of F. graminearum. 【Method】 According to the budding yeast carbon source regulation factorMig1gene sequence on SGD and NCBI databases, the carbon source regulation factor FgCreA in F. graminearum was determined. the sequences of FgCreA orthologs of other species were retrieved from NCBI database. multiple sequence alignment of CreA orthologs was performed with ClustalW2 software, and phylogenetic tree was constructed by MEGA5 software to determine their evolutionary relationships. The protein properties of FgCreA were predicted via InterProScan. The promotor regions of the carbon absorption structure genes and DON biosynthesis genes were obtained from the NCBI database, then the potential FgCreA binding sites in these promotor regions were analyzed. Primer5 software was used to design primers, the FgCREA gene knockout construct was generated by Split-PCR and then introduced into the wild type strain PH-1 by PEG-mediated protoplast transformation. PCR and Southern blot were used to confirm the FgCREA gene deletion mutants (Fgcrea). The function of FgCREA was analyzed according to the vegetative growth, sexual reproduction and pathogenicity of the Fgcrea mutants. 【Result】There is only one carbon metabolism regulator gene FgCREA (FGSG_09715) in F. graminearum. The 416-amino acid protein encoded by FgCREA has two conserved C2H2 zinc finger regions. FgCreA homologue proteins have a certain degree of homology and are highly conserved in fungi. The promoter regions of the carbon absorption structure genes (XYL2, ARA1, ICL1, PG1, SUC2) and DON biosynthesis genes (TRI1, TRI3, TRI4, TRI5, TRI6, TRI7, TRI8, TRI10, TRI12, TRI101) contained FgCreA DNA binding sites. Two FgCREA gene knockout mutants were obtained and screened by PCR and Southern blot. The growth rate of Fgcrea mutants was 90% lower than that of wild type. The conidia morphology of mutants was normal, but the conidiation decreased by 88% compared with the wild type. In the sexual reproduction stage, the Fgcrea mutants could produce normal perithecia, ascus and ascospore, but it delayed 20-28 d longer than the wild type. The Fgcrea mutants were sensitive to salt stress, and were almost non-pathogenic on wheat. 【Conclusion】The carbon metabolic regulator FgCreA plays important roles in vegetative growth and sexual reproduction, and is essential for plant infection in F. graminearum. However, whether FgCreA affects transcription of the carbon absorption structure genes and DON biosynthesis genes remains to be verified.

Heterogeneity Analysis of Ralstonia solanacearum Mutants by Tn5 Transposon Using High Performance Ion-Exchange Chromatography

ZHENG XueFang, LIU Bo, ZHU YuJing, CHEN DeJu, CHEN XiaoQiang

Scientia Agricultura Sinica. 2018, 51(2):  268-278.  doi:10.3864/j.issn.0578-1752.2018.02.007

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【Objective】The objective of this study is to analyze the heterogeneity of Ralstonia solanacearum mutants by Tn5 transposon and screen out an avirulent mutant with high control efficiency. 【Method】 The pathogenicity of sixty R. solanacearum Tn5 transposon mutants was identified by using the attenuation index (AI, a quantitative index for calculating the pathogenicity of R. solanacearum) and tomato plant inoculation bioassay. The chromatographic behavior heterogeneity of different pathogenic mutants of R. solanacearum was analyzed by using high performance ion-exchange chromatography. A chromatography titer index (CTI_i) was expressed as CTI_i=S_i/ (S₁+S₂+S₃)×100% (i=1, 2 or 3; S₁, S₂ and S₃ represent peak areas of P₁, P₂ and P₃, respectively), and the relationship of CTI_i with the pathogenicity and control efficiency of tested mutants was also analyzed based on Pearson correlation coefficient (PCC). 【Result】 Based on the value of AI and the results of bioassay, sixty R. solanacearum mutants were divided into three pathogenic types: virulent, avirulent and interim. Thirty-three mutants belonged to virulent mutant with the value of AI ranged from 0.49 to 0.63 and disease incidence ranged from 66.33% to 100% (15 days after inoculation). Twenty mutants were avirulent with the value of AI ranged from 0.78 to 0.89 and the disease incidence was 0. There were only seven interim mutants with the value of AI ranged from 0.68 to 0.73 and disease incidence ranged from 24.17% to 45.92%. The control efficiency of twenty avirulent mutants against tomato bacterial wilt disease ranged from 16.68% to 92.45%. The best control efficiency was obtained by T831 of 91.74%, followed by T780 (87.51%). The mutant of T497 had the worst control efficiency of 16.68%. Using high performance ion-exchange chromatography, R. solanacearum mutants were successfully separated into three chromatographic peaks: P₁ (retention time of 0.6 min), P₂ (retention time of 4.4 or 4.5 min) and P₃ (retention time of 5.9 or 6.0 min). Both virulent and avirulent mutants had three different peaks: single peak, double and three peaks. However, the interim mutants only had double  peaks and three peaks types. For virulent mutants, the intense peak is P₃, while for avirulent mutants, P₁ is the majority. The virulent mutants with CTI₃ of 100% had high pathogenicity, which could induce tomato plant bacterial wilt with disease incidence up to 85%. The virulent mutants with CTI₃ less than 100% had the disease incidence ranged from 66.33% to 84.14%. The avirulent mutants with CTI₁ of 100% had high control efficiency which reached up to 80%, and the avirulent mutants with CTI₁ less than 90% had low control efficiency which ranged from 16.68% to 63.62%. The correlations of CTI₃ and the pathogenicity of virulent mutants were significantly positive (P＜0.01), with PCC of 0.62. The relationships between CTI₁ and control efficiency against bacterial wilt disease of the avirulent mutants were also significantly positive (P＜0.01), with PCC of 0.80. 【Conclusion】 R. solanacearum mutants by Tn5 transposon have three pathogenic types, which resulted in different chromatographic behaviors. CTI₁ constructed in this study can be used for rapidly selecting an avirulent mutant with high control efficiency and CTI₃ can be used for calculating the pathogenicity of R. solanacearum.

Distribution and Genetic Diversity of Barley yellow striate mosaic virus in Northern China

YANG Fei, ZHANG AiHong, MENG FanSi, HUO LiangZhan, LI XiWang, DI DianPing, MIAO HongQin

Scientia Agricultura Sinica. 2018, 51(2):  279-289.  doi:10.3864/j.issn.0578-1752.2018.02.008

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【Objective】 The objective of this study is to clarify the distributionand genetic diversity of Barley yellow striate mosaic virus (BYSMV) in major wheat production areas in northern China, and to provide a theoretical basis for the early warning, prevention and control of epidemic diseases. 【Method】During 2008-2016, about 864 suspected virus-infected samples were collected from 66 districts in Hebei, Shandong, Jiangsu, Anhui, Henan, Shaanxi and Shanxi provinces, all the three viruses including BYSMV, Rice black-streaked dwarf virus (RBSDV) and Northern cereal mosaic virus (NCMV) were identified using one-step multiplex RT-PCR. L and N gene fragments of BYSMV were obtained by RT-PCR amplification and cloned, and then determined by nucleotide sequence analysis. The sequences were analyzed by softwares of MEGA, DnaSP, and PAML to elucidate the phylogenesis and genetic diversity of BYSMV isolates. 【Result】 A total of 336 samples collected from 48 districts in 7 provinces were detected with BYSMV and the detection rate was 38.89%. The virus was mainly distributed in Shaanxi, Hebei, Shanxi and Shandong. In addition, it was also distributed in Henan and northern Anhui. Xuzhou and Pizhou in Jiangsu Province were only localized. Phylogenetic analysis showed that the population could be divided into two subgroups (I and II) based on fragments of L and N genes. The isolates in subgroup I were derived from all 7 provinces, but isolates in subgroup II were only from Shaanxi and Shanxi provinces. It was indicated that Iran isolate was related to the subgroup II isolates based on the phylogenesis of L gene sequence. The clustering of the isolates was related to their geographical origins, and not to the host plants or sampling dates. Genetic analysis by using 7 softwares of the RDP package showed that there was no evidence supported for the recombination. Selection stress analyses showed that the ω (dN/dS) values varied from 0.02 to 0.19 which were far less than 1 within or between subgroups. It was indicated that the population was undergoing purifying selection. Haplotype diversity (Hd) values (0.90909 and 0.99524) of L and N gene fragments were greater than 0.5 and nucleotide diversity (π) values (0.01324 and 0.01224) were higher than 0.005, indicating that there was a high level of genetic diversity in the population of BYSMV in China. genetic differentiation based on L and N gene fragments showed that the fixation indices F_ST (0.32201 and 0.37326) of eastern and western subpopulations were greater than 0.25. The difference of statistical test was significant, which indicated that the BYSMV population in eastern and western regions was seriously differentiated. The Nm values (0.53 and 0.42) were less than 1, which indicated that the limited gene flow was the main reason of genetic differentiation.【Conclusion】 BYSMV is widely distributed in wheat producing areas of northern China, and occurred in Hebei, Shandong, Jiangsu, Anhui, Henan, Shaanxi and Shanxi provinces on different levels. The population of BYSMV had a high level of genetic diversity in China, and there is a severe genetic differentiation between the eastern and western subpopulations.

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

Effects of Long-Term Different Fertilization and Irrigation Managements on Soil Bacterial Abundance, Diversity and Composition

YANG YaDong, WANG ZhiMin, ZENG ZhaoHai

Scientia Agricultura Sinica. 2018, 51(2):  290-301.  doi:10.3864/j.issn.0578-1752.2018.02.009

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【Objective】This study was carried out to investigate the bacterial community in a wheat-maize rotation field during wheat growing season in northern China. The effects of long-term different fertilization and irrigation regimes on the abundance, diversity and composition of bacterial community were identified. It will provide evidences for further optimizing fertilization and irrigation managements, improving soil productivity, and maintaining soil microbial diversity.【Method】Based on a long-term fertilization and irrigation experiment carried out in Wuqiao Experimental Station of China Agricultural University, six different soil samples were collected after wheat was harvested, including chemical fertilization and no irrigation (CI0), chemical fertilization and irrigation at the jointing stage (CI1), chemical fertilization and irrigation at the jointing and filling stages (CI2), manure fertilization and no irrigation (MI0), manure fertilization and irrigation at the jointing stage (MI1), and manure fertilization and irrigation at the jointing and filling stages (MI2). The abundance, diversity and composition of the bacteria community in different soil samples were revealed by using real-time PCR and Illumina Miseq sequencing platform, targeting the 16S rRNA gene. Correlation analysis was carried out between soil properties and the bacteria community abundance, diversity and structure.【Result】Irrigation significantly increased soil moisture and soil pH, and manure fertilization significantly increased Total Organic Carbon (TOC) content compared with no irrigation and chemical fertilization treatments, respectively. The bacterial 16S rRNA gene copy numbers ranged from 4.34×10⁹ to 1.39×10¹⁰ g^-1 d.w.s in different treatments. Irrigation significantly increased the bacterial 16S rRNA gene copy number by 1.17-1.60 and 0.76-1.93 times in the chemical and manure fertilization treatments, respectively. The bacterial α diversity index results showed that irrigation but not fertilization significantly affected bacterial α diversity index. At the phylum level, thirty-nine phyla were obtained in all the treatments, among which Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria and Bacteroidetes were the predominant phyla, accounting for 77.22%-86.28% of the total reads. There were significant variations in relative abundance of Actinobacteria【Conclusion】Irrigation greatly altered the abundance, diversity, and structure of the bacterial community, while fertilization had a minor effect on the abundance and structure of the bacterial community, and soil moisture and soil pH were the potential environmental factors associated with the bacterial community variations. (11.09%-27.01%), Bacteroidetes (5.45%-12.13%) and Saccharibacteria (2.41%-3.77%) among different treatments. Irrigation significantly decreased the relative abundance of Actinobacteria and Saccharibacteria by 36.48%-48.03%, 22.17%-33.67% and 15.21%-45.54%, 13.40%-23.97% in the chemical and manure fertilization treatments, respectively. Hierarchical clustering and principal component analysis (PCA) results showed that both fertilization and irrigation managements affect the bacterial structure, and irrigation had a stronger effect than fertilization on the bacterial structure. In addition, there were significant correlations between soil moisture, soil pH, Total Nitrogen (TN) content, and TOC content and the abundance, α diversity index and structure of the bacterial community.

Spatiotemporal Variation of Water Use Efficiency and Its Driving Forces on the Loess Plateau During 2000-2014

LIU XianFeng, HU BaoYi, REN ZhiYuan

Scientia Agricultura Sinica. 2018, 51(2):  302-314.  doi:10.3864/j.issn.0578-1752.2018.02.010

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【Objective】Clarifying the spatiotemporal changes of water use efficiency (WUE) and its driving forces in the vegetation ecosystem over the Loess Plateau (LP) provide the basis for greater understanding the interaction of ecosystem and hydrology on the LP under the dual disturbance of ecological restoration and climate change.【Method】Using trend analysis and stepwise regression analysis, our study investigates spatiotemporal variation in WUE of vegetation ecosystem and its driving forces on the LP during the period 2000-2014. 【Result】The WUE reveals a significant increasing trend on the LP during 2000-2014 (linear tendency 0.02 gC·kg^-1 H₂O·a^-1, P<0.001). Intra-annual analysis shows that the WUE of different vegetation ecosystems mainly showed a bimodal pattern, which the two peaks appeared in April to May and September to October, respectively. Spatially, the WUE of the LP generally reveals an increasing trend, and the area of increasing trend and significantly increasing trend (P<0.05) accounting for 95.04% and 66.96% of the total study area, respectively, but the patterns of trend in different seasons vary significantly. The mean and trend of WUE among different vegetation ecosystems also vary significantly, among which the mean of WUE of the open shrublands and grasslands are lower than that of other vegetation ecosystems, while the trend of WUE of needleleaf forest decrease significantly. The results of slope statistic show that the WUE of vegetation ecosystem reveals a continuous increase trend in the range of 25°-50°. When annual evapotranspiration (ET) is below 3 700 mm, Gross Primary Productivity (GPP) is positive correlated with ET increase, while when annual ET above 3 700 mm, GPP decrease with the increase of ET. In addition, the WUE of the central and eastern part of the study area is mainly controlled by GPP (57.25%), while ET (42.75%) mainly affects the WUE of the western part of the study area. Moreover, WUE is significantly positive correlated with Leaf Area Index (LAI), indicating the increase of LAI will promote increase in WUE. Stepwise regression analysis shows that precipitation, sunshine duration, and relative humidity are the main climatic factors that lead to the change of WUE and its components in recently 15 years. 【Conclusion】Under the dual disturbance of climate change and anthropogenic activities, the WUE of the LP shows a significantly increasing trend in the past 15 years, and most of the vegetation types show a bimodal pattern within the year. Meanwhile, the ecological restoration project not only improve the vegetation coverage of the LP, but also significantly improve the WUE of vegetation ecosystem of the LP, becoming the main driving forces for explaining changes in WUE on the LP during recent 15 years.

HORTICULTURE

Different SlU6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato

PU Yan, LIU Chao, LI Ji-Yang, AERZU GULI·TaShi, HU Yan, LIU XiaoDong

Scientia Agricultura Sinica. 2018, 51(2):  315-326.  doi:10.3864/j.issn.0578-1752.2018.02.011

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【Objective】U6 promoter is a vital element for the transcription of sgRNA in the CRISPR/Cas9 system. It is necessary to clone some endogenous U6 promoters with high transcription activity and construct CRISPR/Cas9 vector, which could provide a strong technical basis for functional genomics and molecular breeding in tomato. 【Method】 Four different tomato U6 promoters were cloned by first round of PCR amplification from tomato cultivar Zhongshu 4. Each U6 promoter with two different length was truncated and used to construct plant expression vector carried SlU6 promoter::GUS, respectively. The Eight GUS fusion expression vectors were transformed into tomato leaves by agroinfiltration. According to the degree of GUS staining, the promoter with high transcription activity was selected to construct the CRISPR/Cas9 gene editing vector with target sequence from powdery mildew-related gene MLO1 and EDR1. These gene editing vectors were transformed into tomato protoplast by PEG method. The mutation of endogenous target genes in each transformed tomato protoplast was analyzed by a restriction enzyme PCR (RE-PCR) assay. Finally, the types of endogenous gene mutation were analyzed by sequencing. The efficiency of the CRISPR/Cas9 system based on tomato endogenous U6 promoter was verified by the frequency distribution map of mutant loci. 【Result】4 kinds and 8 different lengths of tomato U6 promoters were obtained by two rounds of PCR. Their length was 452, 202, 448, 206, 433, 190, 448 and 218 bp, respectively. After sequences analysis, results showed that the four tomato U6 promoters also contained the USE motif and TATA box which were found in Arabidopsis U6 promoters. The construction of GUS fusion expression vectors driven by corresponding truncated tomato U6 promoters were done and transformed into tomato leaves. The GUS histochemical staining showed that the transformed tomato leaves were dyed blue, which indicated that all 8 SlU6 promoters have transcription activity.The SlU6-2P4 promoter was chose to drive sgRNA transcription and construct the CRISPR/Cas9 system with target sequence from MLO1 and EDR1 respectively. The result showed that endogenous SlU6-2P4 promoter could drive sgRNA transcription and gene MLO1 was edited successfully in tomato. Sequence analysis revealed that all types of gene mutations are base substitution and the hotspot of mutation only exists in the target region of endogenous gene. 【Conclusion】4 kinds of SlU6 promoters with high transcription efficiency were obtained from tomato. The established CRISPR/Cas9 system based on SlU6-2 promoter could successfully achieve the editing of endogenous genes in tomato.

Genome-Wide Identification and Expression Analysis of Auxin Response Factor (ARF) Gene Family in Pear

OU ChunQing, JIANG ShuLing, WANG Fei, ZHAO YaNan

Scientia Agricultura Sinica. 2018, 51(2):  327-340.  doi:10.3864/j.issn.0578-1752.2018.02.012

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【Objective】The objectives of this research are to identify the auxin response factor (ARF) family genes from pear (Pyrus bretschneideri) genome, to know the profile of ARF family such as gene number, gene structure and tissue expression in pear, and to provide theoretical basis for revealing what roles the ARF play in auxin signaling pathway and in growth and development of dwarf pear.【Method】ARF genes in pear genome were identified by BLAST software based on ARF genes from apple and Arabidopsis. SMART, PROSITE, WebLogo 3, DNAMAN 5, MEME, GSDS 2 and MEGA 5.1 software were used for bioinformatics analysis of ARF protein and gene sequences. The qPCR method was used to detect the relative expression of ARF genes in different tissues of dwarf pear ‘Zhongai 1’ and in xylem and phloem of 3 pear cultivars with different growth vigor.【Result】Total of 31 ARF genes were identified from pear genome. All the PbARFs contain two domains of Auxin_resp and B3, and except for PbARF11, 12, 24, 25 and 26, the rest also contain a PB1 domain. Conservative motif analysis result showed that there are 15 motifs in PbARFs, but not every PbARF protein contains all the motifs. The PbARFs were divided into four classes based on phylogenetic analysis. Gene structure analysis result showed that there are 2-15 exons in PbARFs, The gene structure of PbARF is high conservative. The qPCR result showed that all the PbARF genes were expressed in the root, phloem, xylem, leaf, flower and fruit of ‘Zhongai 1’ pear and the expression pattern was various. The relative expression of PbARF29 in the phloem of 3 pear cultivars showed that the more dwarf the plant, the higher the expression, and the relative expression of PbARF16, 17, 18, 27 in the xylem of 3 pear cultivars showed that the more dwarf the plant, the lower the expression.【Conclusion】Auxin response factor family in pear contains 31 genes. All the 31 PbARF proteins contain both of Auxin_resp and B3 conservative domains, and were divided into four classes. The gene structure of PbARF is high conservative. All the 31 PbARFs were expressed in different tissues of ‘Zhongai 1’, root of rootstock Pyrus betulifolia and in phloem and xylem of 3 cultivars. Thereinto, the expression of PbARF29, 16, 17, 18, 27 may be relevant to pear plant height.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Effect of Weaning Liquid Diet at Different Level of Creep Feed Intake on Growth and Development of Lambs

CHAI JianMin, WANG Bo, QI MinLi, WANG ShiQin, TU Yan, TAO XiaoJing, DIAO QiYu, ZHANG NaiFeng

Scientia Agricultura Sinica. 2018, 51(2):  341-350.  doi:10.3864/j.issn.0578-1752.2018.02.013

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【Objective】 This study was conducted to investigate the effects of weaning liquid diet (milk replacer) at different level of creep feed intake on growth performance, slaughter performance and development of visceral organ and gastrointestinal tract of lambs to confirm the best time to wean milk replacer and providing theoretical basis of growth and development of lambs. 【Method】 Lambs were divided into 4 treatments by using single factor experimental design that experimental factor was creep feed intake on the time of weaning milk replacer. Each treatment had 16 lambs and each lamb was as a replication. Sixty-four purebred Hu lambs with similar body weight were selected, weaned breast-milk and fed milk replacer at 10 days of age. Lambs were provided creep feed at 15 days of age and could take the feed freely. When consuming level of dry matter of creep feed reached 200, 300, 400 and 500g, lambs were weaned milk replacer respectively and assigned to A, B, C and D groups correspondingly. During the animal feeding trial, growth performance including body weight, average daily gain, feed intake and feed conversion efficiency was determined. Slaughter experiment was conducted at 90 ages of age to determine slaughter performance including development of visceral organ and gastrointestinal tract of lambs. 【Result】 The results showed as follows: (1) average daily gain in B and C groups was significantly higher than that in D group (P＜0.05) from 10 to 90 days; intake of milk replacer in A and B groups was significantly lower than that in C and D groups (P＜0.05), and A group had lower milk replacer intake than B group (P＜0.05); the creep feed intake in A and B group was higher than that in C and D group (P＜0.05) during the whole trial; the feed conversion efficiency in B and C groups was lower that in D group (P＜0.05) from 10 to 90 d and from weaning milk replacer time to 90 d. (2) rumen weight in A, B and C group was significant higher than that in D group (P＜0.05), and C group was higher than A group (P＜0.05); despite the ratio of rumen to total stomachus compositus weight and live weight before slaughter showed no significance (P＞0.05), the mean values in B and C groups of these 2 parameters were higher than that in A and D group; the weight of reticulum, omasum in C group was higher than that in A and D groups (P＜0.05); the weight of whole stomach in C group was higher than that in A and D groups (P＜0.05), and the ratio of whole stomach weight to live weight before slaughter were not significantly different among 4 groups (P＞0.05), but the mean values of it in B and C groups were higher than those in A and D group; although small intestine weight and the ratio of its weight to live weight before slaughter were not significantly different (P＞0.05) among different groups, the mean values in B and C groups were higher than those in A and D groups; A and B groups had higher large intestine weight than D group (P＜0.05), and no difference was observed in the ratio of large intestine weight to live weight before slaughter among 4 groups (P＞0.05), but the mean values of it were greater in B and C groups than those in A and D groups. (3) B and C group had significant higher heart weight than D group (P＜0.05) while weight of other visceral organs and ratio of their weight to live weight before slaughter were not significantly different (P＞0.05); (4) at 90 days of age, the slaughter body weight, empty body weight, carcass weight and 24h carcass weight in A, B and C groups were higher than those in D group (P＜0.05), and empty body weight in C group was higher than that in A and B groups (P＜0.05). carcass weight and 24h carcass weight in C group were higher than that in A group (P＜0.05); eye muscle area in B and C groups was significantly higher than that in A and D groups (P＜0.05).【Conclusion】 In conclusion, lambs showed better growth and slaughter performance as well as gastrointestinal development when they could consume an equivalent of 300 or 400g dry matter of creep feed for 3 consecutive days and were weaned milk replacer. Therefore, optimal effect could be reached for the Hu lambs to be weaned milk replacer at creep feed intake level of 300g dry matter.

Profiles of miRNAs and Target Gene Analysis with White and Black Skin Tissues of the Tibetan Sheep

WU ZhenYang, TANG XiaoHui, FU YuHua, WANG Sheng, ZHANG Cheng, LI JingJin, YU Mei, DU XiaoYong

Scientia Agricultura Sinica. 2018, 51(2):  351-362.  doi:10.3864/j.issn.0578-1752.2018.02.014

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【Objective】 Tibetan sheep is one of three coarse wool sheep breeds in China, distributing mainly in Tibet and its adjacent alpine pastoral areas, such as Qinghai, Gansu, Sichuan, Yunnan. The wool of Tibetan sheep, felt smooth and warm duo to the long and thick fibers, is good raw material for manufacture of Tibetan carpet. Coat color is an important economic traits. However, at present, the mechanism of molecular regulation of sheep coat color is not clear. In this study, the transcriptome of skin tissues of different color (black and white) was sequenced with a aim to explore the role of miRNAs at post-transcriptional levels in different coat color skin tissues and possible regulatory pathways. 【Method】Healthy sheep with black and white coat color was sacrificed to provide skin tissues for RNA extraction and miRNA analysis. Through miRNA sequencing and bioinformatics analysis, miRNA expression profiles of skin tissues of black and white wool color were obtained. Then the differentially expressed miRNAs were screened and the related target genes were predicted. Through the target gene analysis, the signal pathway which is related to Tibetan coat color skin traits were proposed. 【Result】 A total of 85.76 million original reads and 85.08 million clean reads were obtained from the analyzed tissues. Among the clean reads were 334 known miRNAs and 59 newly identified miRNAs and 23 of them were differentially expressed between white and black color tissues. In the 23 differentially expressed miRNAs, 14 and 9 were up-regulated and down-regulated in white skin tissues respectively. miR-2284b and miR-744 were the type that is expressed only in white Tibetan sheep skin, and miR-23b, miR-411a-5p, miR-30c, miR-423-3p, miR-324-5p were the miRNAs that expressed only in black skin. Among them, miR-10a can participate in the regulation of a variety of signaling pathways, involving proliferation, differentiation, apoptosis, cell adhesion. MiR-23b is associated with Wnt, Notch and other signal pathways, which may related to melanin production. MiR-411a-5p, miR103 and miR-200b may also closely relate to regulation of hair color. A total of 981 target genes were predicted to be involved in coat color control, most of which were related to WNT signaling pathway, MAPK pathway, EDNRB signaling pathway and Melanogenesis pathway. The melanogenesis pathway was found to be associated with the WNT signal pathway, the KIT signal pathway, and the EDNRB signal pathway. The synthesis of melanin may be regulated by downstream TYR gene (tyrosinase gene) through MITF gene. Quantitative analysis showed that five miRNAs were up-regulated in black skin tissue, and two miRNAs were up-regulated in white tissues. The quantitative results were consistent with the sequencing results. 【Conclusion】 The miRNA expression profile of the skin of Tibetan sheep were obtained and the miRNAs and signal pathways which may be related to control of coat color were obtained by bioinformatics analysis. These results indicated that the color traits could be regulated by many miRNAs and involved many signal pathways. This may help understand the level of post-transcriptional regulation of the process, and lay the foundation for further functional verification.

Current Status of Transgenic Technologies for Safety Consideration in Silkworm (Bombyx mori) and Future Perspectives

LONG DingPei, HAO ZhanZhang, XIANG ZhongHuai, ZHAO AiChun

Scientia Agricultura Sinica. 2018, 51(2):  363-373.  doi:10.3864/j.issn.0578-1752.2018.02.015

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Transgenic technology is an important tool for gene function analysis and genetic improvement of biological variety.At present, the safety problems of GMOs mainly focus on the safety of genetic manipulation techniques and genetically modified products. In recent years, the researches on the safety of GMOs werefocused on evaluating the security level of genetically modified crops, aquatic animals and poultry, livestock and other large animals in medicine, agriculture, and food industry, but the reports of the safety of genetic manipulation techniquesare still rare in the agricultural insects which have important scientific and economic value, and the Ministry of Agriculture has not yet set a precedent for the safety assessment of transgenic insects. The silkworm (Bombyx mori) is an important mode lepidopteran and economic insect for agriculture. In-depth research on the safety of transgenic technologies of B. mori, which has important value and significance for promoting the development of basic genetics and silk industry. Since the first report of the germline transformation of the B. mori using a piggyBac transposon-derived vector in 2000, transgenic technology has been widely used in the basic research of gene function identification and the applied research of creating new varieties with specific gene differences in B. mori. But the safety problems of transgenic B. mori have become the key problems that hindering the practical application of transgenic B. mori. Therefore, doing research on the safety of transgenic technologies of B. mori is of great significance to promote the safety assessment and industrialization of transgenic B. mori. This paper summarizes the establishment and research status of the conditional gene targeting-based safety of transgenic technologies of B. mori, while the development trends and application prospect of these technologies in B. mori are also discussed, as well as provide references for establishing and perfecting the safety of transgenic technologies in other transgenic animals, especially the agriculture insect species.

RESEARCH NOTES

Dry Matter Accumulation and Water Use Performance of Winter Wheat Cultivar Zhongmai 175 Under Three Limited Irrigation Levels

LI FaJi, XU XueXin, HE ZhongHu, XIAO YongGui, CHEN XinMin, WANG ZhiMin

Scientia Agricultura Sinica. 2018, 51(2):  374-385.  doi:10.3864/j.issn.0578-1752.2018.02.016

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【Objective】 Zhongmai 175 is a widely adapted and largely planted winter wheat cultivar both in irrigated areas of northern winter wheat zone and rainfed areas of Huang-Huai Rivers Valley wheat zone. Therefore, understanding its dry matter accumulation and water use performance could be helpful to reveal water saving and high-yielding mechanism of wheat.【Method】Field experiments were conducted in Wuqiao of Hebei province and in Shunyi of Beijing with two wheat cultivars (namely, Zhongmai 175 and Jingdong 17) under three limited irrigation levels, including no irrigation in whole growing stage (W0), irrigation in jointing stage (W1, 75 mm), and irrigations in jointing and flowering stage (W2, 75 mm +75 mm). Phenotypic traits such as population vitality, dry matter accumulation and distribution, yield and water use efficiency (WUE) were compared to understand the differences between the two cultivars as well as their responses to different irrigation levels.【Result】The highest yields were obtained under W2 treatment for both cultivars, while yield decreased with the reduction of irrigation. The W0 treatment mainly reduced the kernel number per square meter due to the remarkable decrease of 47-67 in spike number per square meter (SN) and 1.6-5.1 decrease in kernel number per spike (KNS). The W1 treatment mainly reduced the thousand-kernel weight (TKW) about 0.6-1.5 g. Water deficit notably reduced evapotranspiration (ET) and population biomass. Nevertheless, it remarkably increased the translocation amount (TA) of dry matter accumulated before flowering to grains. However, moderate water deficit (W1) increased the WUE of both cultivars. Compared with Jingdong 17, Zhongmai 175 showed higher yield and yield stability under different irrigation treatments. In Zhongmai 175, most of the physiological traits like normalized difference vegetation index (NDVI), canopy temperature depression (CTD) and content of conserved water-soluble carbohydrates (WSC) in stem in middle grain-filling stage were higher; contrastingly, water sensitive coefficient (WS) of most yield traits was lower in Zhongmai 175. These characters might be the physiological basis for the high yield and efficiency of Zhongmai 175. The WS of KNS and biomass showed close correlation with WS of yield, as well as WS of NDVI and CTD in early stage of grain-filling.【Conclusion】The rapid accumulation of dry matter in early stage, large pool capacity and strong ability of population vitality might be the main reasons for the water-saving and high yield of Zhongmai 175. The differences of NDVI and CTD in early stage of grain-filling under different irrigation treatments could be used as fast and comprehensive evaluation parameters in detecting water sensitivity of different cultivars.

Wheat Yield and Growing Period in Response to Field Warming in Different Climatic Zones in China

GAO MeiLing, ZAHNG XuBo, SUN ZhiGang, SUN Nan, LI ShiJi, GAO YongHua, ZHANG ChongYu

Scientia Agricultura Sinica. 2018, 51(2):  386-400.  doi:10.3864/j.issn.0578-1752.2018.02.017

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【Objective】Global warming has been recognized as a key impact factor for wheat growth and development. However, the responses of wheat growth and development to warming are still remain unclear, and have not been systemically quantified in different climate regions of main wheat producing area in China. Therefore, there is a special need to systematically quantify the magnitude and mechanisms of field warming impacts on wheat yield and growing period at different periods in a day and the main climatic regions. 【Method】This study collected 21 published literatures between 1990-2017 from nationwide with the effects of field warming on wheat yield and development. In addition, the Meta-analysis was used to systemically quantify the magnitude of field warming during entire wheat growing season on wheat yield and growing period at different climate regions. 【Result】 The results indicated that: (1) Field warming (0-3 °C) significantly increased the wheat yield, thousand kernel weight, and grain number per spike under subtropical monsoon climate whose the average growth rates were 8.2%, 6.3%, and 4.7%, respectively, and significantly increased the wheat yield, spike numbers, and grain number per spike under temperate monsoon climate whose the average growth rates were 6.8%, 3.9% and 5.5%; By contrary, field warming (0-3 °C) reduced the wheat yield, thousand kernel weight and grain number per spike under temperate continental climate whose the average change rates were 10.2%, 5.9%, and 8.3%, respectively. Specifically, the wheat yield significantly were increased (8.5%) by 0-2 °C of field warming and were not significantly changed by 2-3 °C of field warming under subtropical monsoon climate; The increment of wheat yield by 2-3 °C of field warming was 14.5% under temperate monsoon climate; On the contrary, wheat yield were significantly reduced by 10.1% and 15.9% by 0-2 °C and 2-3 °C of field warming under temperate continental climate, respectively. (2) The entire duration of wheat growing period was shorten by 3.3% and 7.1% by field warming (0-3 °C) under subtropical monsoon climate and temperate monsoon climate, but was not changed apparently under temperate continental climate. At the same time, the duration of wheat reproductive period in temperate monsoon climate and temperate continental climate were not changed significantly, while the duration of reproductive growth in subtropical monsoon climate was increased significantly (8.7%). (3) On the whole, though the effects of warming period in a day on wheat yield and development were varied among different climatic regions, the wheat yield were significantly increased by 10.5% and 15.0% under 0-2 °C and 2-3 °C of night warming within all climatic regions. 【Conclusion】 The effect magnitude of field warming on wheat yield and growing period was varied under different climatic regions and the period in a day. The findings of this study could provide scientific base for rational optimization and arrangement of cropping system within the main producing areas in China under new climate change situations.