Scientia Agricultura Sinica

Cloning, Expression and Functional Analysis of a Male Fertility Gene ThMs1 in Bread Wheat

HENG YanFang,LI Jian,WANG Zheng,CHEN Zhuo,HE Hang,DENG XingWang,MA LiGeng

Scientia Agricultura Sinica. 2020, 53(23): 4727-4737. doi:10.3864/j.issn.0578-1752.2020.23.001

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【Objective】To clone a new male fertility gene, ThMs1, from the ms1g-4Ag wheat-Thinopyrum ponticum alien addition line, and characterize the expression pattern and biological function of ThMs1 in bread wheat, with the hope that ThMs1 will be useful in the application in the preparation of wheat hybrid breeding system. 【Method】4Ag chromosome from Thinopyrum ponticum in wheat-Thinopyrum ponticum alien addition line was identified by GISH. ThMs1 was cloned from the wheat-Thinopyrum ponticum alien addition line by homology-based cloning, and the analysis of the function of ThMs1 in the control of wheat male fertility was performed through stable genetic transformation. RT-PCR and qRT-PCR were performed to detect the expression pattern of ThMs1, further analyzing its tissue-specific expression by RNA in situ hybridization. The expression of ThMs1 protein in vivo was detected by Western blot using specific anti-Ms1 antibodies. ThMs1 protein structure were predicted by SignalP software. The detection of lipid-binding activity of ThMs1 was performed by dot blot. 【Result】The wheat-Thinopyrum ponticum alien addition line harbored 4Ag chromosome from Thinopyrum ponticum. ThMs1, the homolog of bread wheat male fertility gene Ms1, was cloned from the wheat-Thinopyrum ponticum alien addition line. The functional complementation analysis confirmed that the ThMs1 can completely restore the male fertility phenotype of the wheat ms1. The phylogenetic analysis suggested that the MS1 only exists in the Poaceae family. RT-PCR and qRT-PCR analysis confirmed that ThMs1 gene was expressed in anthers during meiosis. RNA in situ hybridization analysis revealed that ThMs1 was specifically expressed in microspore during wheat microsporogenesis. Western blot showed that ThMs1 was detected in wheat anther. The structure prediction analysis revealed that it has a predicted lipid binding domain, and protein lipid-binding experiments indicated that ThMs1 protein specifically binds PA and several phosphatidylinositols. 【Conclusion】A new male fertility gene, ThMs1, was cloned from wheat-Thinopyrum ponticum alien additiona line. ThMs1 exhibits similar expression pattern to that of Ms1 in bread wheat, and ThMs1 binds to the same phospholipid molecules as Ms1, thus ThMs1 is a structural and functional homolog of wheat Ms1. Therefore, the present work identified a new male fertility gene, and provided a foundation for the establishment of wheat hybrid breeding system through molecular design using wheat ms1.

Identification of ARF Gene Family and Expression Pattern Induced by Auxin in Fagopyrum tataricum

HAO YanRong,DU Wei,HOU SiYu,WANG DongHang,FENG HongMei,HAN YuanHuai,ZHOU MeiLiang,ZHANG KaiXuan,LIU LongLong,WANG JunZhen,LI HongYing,SUN ZhaoXia

Scientia Agricultura Sinica. 2020, 53(23): 4738-4749. doi:10.3864/j.issn.0578-1752.2020.23.002

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【Objective】 The objective of this study is to lay a foundation for the further functional studies and application of ARF genes by identifying the ARF gene family in tartary buckwheat (Fagopyrum tataricum), and analysis of gene structure, conserved domains, phylogeny, tissue expression characteristics, and gene expression levels under exogenous auxin treatment. 【Method】 The data of transcriptome and ARF conservative domains (PF06507) were analyzed to screen for ARF family members in tartary buckwheat. TBtools software was used to analyze the gene structure, NCBI and MEME were used to predict the conserved domain and motif of ARF proteins online, and MEGA X was used to construct ARF protein phylogenetic trees for tartary buckwheat, Arabidopsis thaliana, Oryza sativa, Fagopyrum esculentum, Beta vulgaris, and Glycine max. The FPKM values of six tissues of roots, stems, leaves, flowers, green grains, and black grains were analyzed in the transcriptome data of tartary buckwheat. Heat maps of FtARFs were drawn using TBtools HeatMap to analyze the tissue expression specificity of FtARFs. To predict the cis-acting elements of the FtARFs promoter specifically expressed on the stem, the PlantCARE online website was used. Two parts of tall buckwheat (ZNQ189 and PI673849) and two parts of dwarf buckwheat (PI658429 and PI647612) were treated with 0.5 mg·L ^-1 IAA and the elongation characteristics of the hypocotyls were analyzed. Samples were taken at different time periods (0, 0.5, 1, 6, 12, 24, and 48 h) after auxin treatment, and the expression differences of FtARFs in different hypocotyls were analyzed by qRT-PCR. Moreover, paraffin sections of four materials grown for 7 days were saffron and green stained, and the cell length was measured. 【Result】Total of 26 FtARFs were identified in the buckwheat genome. Chromosome mapping analysis showed that in addition to chromosome 4, FtARFs were distributed in other chromosomes. Analysis of physical and chemical properties showed that amino acid residues ranged from 331 to 1 083 aa and the theoretical isoelectric point ranged from 5.34 to 8.63. Conserved motif analysis identified several differences in motif composition among different groups. Gene structure analysis showed that the number of FtARFs exons ranged from 2 to 15, showing large variation. Phylogenetic analysis showed that 114 ARF proteins were divided into four groups (Group Ⅰ to Group Ⅳ), and FtARFs were distributed throughout all groups. The results of tissue-specific analysis showed that FPKM values of FtARFs differed significantly in different tissues. In roots, stems, and flowers, seven, nine, and four genes showed high expression, respectively, whereas in leaves, green grains, and black grains, the expression values remained low. When exogenous auxin was applied to these four buckwheat materials, the trend of hypocotyl elongation differed, which is consistent with observed changes in cell length. qRT-PCR showed that the expression of FtARFs was higher during the early stage (0.5-1 h) of auxin treatment, and decreased during later stages. When buckwheat seedlings were treated for 0.5 h, the expression of most genes was induced. 【Conclusion】The tartary buckwheat ARF gene structures and protein motifs show diversity among groups and conservation within groups. FtARFs show tissue expression specificity, and nine FtARFs, that are specifically expressed in the stem, respond to IAA induction. These exert a regulatory role in the stem elongation of tartary buckwheat.

Effects of Exogenous ABA on Pre-Harvest Sprouting Resistance and Quality of White and Red Wheat Cultivars

ZHANG Xue,YANG HongKun,ZHENG Ting,XIAO Yun,MO Piao,FAN GaoQiong

Scientia Agricultura Sinica. 2020, 53(23): 4750-4763. doi:10.3864/j.issn.0578-1752.2020.23.003

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【Objective】Pre-harvest sprouting (PHS) resulted from higher rainfall during the harvest period is one of the crucial reasons degrade food uses quality of wheat flour in Southwest wheat production region of China. This study was carried out to investigate the inhibition effect of exogenous abscisic acid (ABA) to pre-harvest sprouting, and the changes in grain quality with respecting to PHS sensitivity was also measured. 【Method】Two cultivators with pre-harvest sprouting resistance (sensitive: white-seeded wheat Zhongkemai 138; insensitive: red-seeded wheat Mianmai 367) were used as experimental materials. The anti-photodegraded ABA (0, 50, and 100 mg·L ^-1) were sprayed at 15 (initial filling stage), 30 (late filling stage) and 35 days (physiological maturity stage) after anthesis (DAA) to investigate the changes in germination traits, α-amylase activity and grain quality. 【Result】 (1) The application of 50-100 mg·L ^-1 ABA at 30 DAA could inhibit PHS, with the optimum spraying time achieved at 30 DAA. In normal years (2018), the wheat spraying with 50 mg·L ^-1ABA was better than other treatments as compared to the control, the germination rate of Zhongkemai 138 was reduced by 13.8 and 3.8 percentage points at physiological maturity and dough stage, respectively, and the PHS sensitivity cultivator (Mianmai 367)was decreased by 23.5 and 9.7 percentage points, respectively, as compared with control. In 2019 (rainy season), spraying with 100 mg·L ^-1 ABA performed better than other treatments with the germination rate of Zhongkemai 138 was reduced by 22.5 and 19.6 percentage points during the physiological maturity and dough stage, respectively, as compared with control, and PHS sensitivity cultivator (Mianmai 367) was reduced by 10.0 and 12.0 percentage points than that of control, respectively. Meanwhile, the inhibitory effects of ABA were all released at 60 days after harvest, and did not affect subsequent seed germination. (2) Exogenous application of ABA could reduce α-amylase activity and inhibited α-amylase activity, which further delayed the hydrolysis of starch in 35-45 DAA. Compared with the control, the α-amylase activity and soluble sugar content were decreased by 30.1%, and 41.9%, respectively, and the starch content was 10.2 percentage points higher when the 100 mg·L ^-1 ABA was spraying at 30 DAA. (3) The application of 100 mg·L ^-1 ABA improved the precipitation value by 4.3%-8.8%, and exogenous application of ABA showed a greater impact on the starch content with the amylopectin content and total starch content increased by 8.1 and 7.6 percentage points, and the amylose/amylopectin ratio decreased by 18.2%. Further, the pasting properties of flour were also improved with the falling value, peak viscosity and disintegration value was 20.9%-24.2%, 26.5%-51.4% and 12.4%-43.4% higher than that of CK, respectively. 【Conclusion】The application of 50-100 mg·L ^-1 ABA at 30 DAA could effectively reduce pre-harvest sprouting by inhibiting α-amylase induced starch hydrolysis without decline the protein quality, and thereby improved the pasting properties of wheat flour by enhancing amylopectin and total starch contents, reducing the ratio of amylase and amylopectin. Therefore, the application of 50-100mg·L ^-1 ABA at 30 DAA was highly recommended for farmers to enhance the pre-harvest sprouting resistance and reducing the losses in food uses quality in Southwest wheat production region of China.

Effects of Intercropped Wheat Straw Retention on Canopy Temperature and Photosynthetic Physiological Characteristics of Intercropped Maize Mulched with Plastic During Grain Filling Stage

YIN Wen,CHAI Qiang,YU AiZhong,ZHAO Cai,FAN ZhiLong,HU FaLong,FAN Hong,GUO Yao

Scientia Agricultura Sinica. 2020, 53(23): 4764-4776. doi:10.3864/j.issn.0578-1752.2020.23.004

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【Objective】The photosynthetic and physiological characteristics are important factors affecting the yield of crops. The canopy temperature reflects the energy balance of crops, which is closely related to the photosynthetic physiology and yield formation of the crop. Study on crop canopy temperature, photosynthetic and physiological characteristics, and yield formation under different straw and plastic mulching methods is of great significance for optimizing cropping system and improving crop production potential in arid oasis irrigation areas. 【Method】The field experiment was conducted in Hexi oasis irrigation region from 2014 to 2016, to determine the effects of straw retention and plastic mulching approaches on canopy temperature and photosynthetic physiological characteristics of maize in wheat-maize intercopping. 【Result】Intercropping could reduce the canopy temperature of maize in the grain filling stage, compared to sole cropping. No-tillage with 25 to 30 cm straw mulching in wheat strip and two-year plastic mulching in maize strip (NTSI2) and no-tillage with 25 to 30 cm straw standing in wheat strip and two-year plastic mulching in maize strip (NTSSI2) had significant effect at reducing the canopy temperature of maize, and reduced the canopy temperature of maize by 10.3% and 7.5%, respectively, compared to sole maize with annual new plastic mulching (CTM), which reduced canopy temperature by 7.6% and 4.7%, respectively, compared to conventional tillage without straw retention in wheat strip and annual new plastic mulching in maize strip (CTI). According to canopy-air temperature difference, it could be seen that NTSI2 treatment had less changes in canopy temperature with air temperature during maize filling stage, which could reduce the adverse effect of temperature change on the growth and development of maize. Intercropping could increase the photosynthetic rate (P_n), transpiration rate (T_r) and leaf water use efficiency (WUE_L) during the grian-filling stage of maize, compared to sole cropping. The treatments on NTSI2 and NTSSI2 had greater P_n of maize by 23.0% and 18.1% than that of CTM, and greater P_n by 13.4% and 8.9% than that of CTI, respectively. Also, NTSI2 and NTSSI2 had greater T_r of maize by 7.9% and 5.8% than that of CTM, and greater T_r by 6.1% and 4.1% than that of CTI, respectively. Thus, NTSI2 and NTSSI2 increased WUE_L of maize by 14.4% and 12.0% than that of CTM, and increased WUE_L by 7.2% and 4.9% than that of CTI, respectively. The treatment on NTSI2 had the highest increasing effect on WUE_L, it also had the potential for efficient use of water. Intercropping increased maize grain yield by 52.2% over sole cropping. The grain yield of NTSI2 and NTSSI2 was 57.2% and 53.4% greater than that of CTM treatment, and was 17.6% and 14.7% greater than that of CTI treatment, respectively. 【Conclusion】 No tillage and 25 to 30 cm straw retention combined with two-year plastic film mulching was the most feasible technology for realizing high yield and efficient utilization of resources of wheat-maize intercropping in the oasis irrigation region.

The Genetic Diversity, Pathogenicity, and Toxigenic Chemotypes of Fusarium graminearum Species Complex Causing Maize Ear Rot

WANG BaoBao,GUO Cheng,SUN SuLi,XIA YuSheng,ZHU ZhenDong,DUAN CanXing

Scientia Agricultura Sinica. 2020, 53(23): 4777-4790. doi:10.3864/j.issn.0578-1752.2020.23.005

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【Objective】The objective of this study is to clarify the genetic structure, pathogenicity, toxigenic chemotypes, and their relationship of Fusarium graminearum species complex (FGSC) causing maize ear rot in China, and to provide reference information for prevention and control of Fusarium ear rot of maize. 【Method】The samples were collected from the main maize producing areas in China. Twenty-two pairs of SSR and 10 pairs of VNTR primers, along with TEF-1α, β-tubulin and RPB2 gene sequences were used for genetic diversity and Popgen32, NTsys2.1, and STRUCTURE2.3.4 software were used to analyze the data and construct the phylogenetic tree. The pathogenicity of FGSC was determined using the silk channel injection inoculation method, and the specific primers were used to detect the toxigenic chemotypes. 【Result】A total of 48 alleles were detected by SSR and VNTR primers, 39 polymorphic sites were found, with a polymorphic band ratio of 81.25% among 45 strains, the polymorphic bands ranged from 2 to 4. The average Shannon’s information index and Nei’s genetic diversity index of the 7 FGSC geographic populations were 0.41 and 0.29, respectively, the genetic similarity in 7 regions was 0.6677-0.8797, and the genetic distance was 0.1282-0.4039, indicating that there existed rich genetic diversity among the flora. Based on the Nei’s genetic distance, 7 FGSC geographical populations were divided into 3 groups by UPGMA clustering. The population structure of FGSC stains could be divided into two different groups by STRUCTURE2.3.4. Most of the strains from Northwest China belonged to group A, those in Central China and South China belonged to group B, and more than 50% of the strains in Northeast China belonged to group B. Based on the sequences of TEF-1α, β-tubulin and RPB2, FGSC was composed of F. graminearum, F. asiaticum, F. boothii, and F. meridionale. There were 142 single nucleotide polymorphisms (SNPs) among these Fusarium strains. The dendrogram constructed by these differential sequences could clearly show the genetic differentiation within and between species. The genetic diversity within each Fusarium species was rich. Among four Fusarium species, the pathogenicity of F. graminearum was the strongest, with an average diseased ear area of 20.79%. The average diseased areas of F. asiaticum, F. boothii, and F. meridionale were 15.79%, 11.77%, and 8.12%, respectively. The difference in the percentage of average diseased area was significant between Fusarium species. The toxigenic chemotypes of F. graminearum were 15ADON, 3ADON, NIV, and 3ADON + 15ADON + NIV, F. boothii contained 15ADON, 3ADON, NIV, 3ADON + 15ADON, and 3ADON + 15ADON + NIV chemotypes, the toxigenic chemotypes of F. meridionale were 15ADON, 3ADON, and 15ADON+ NIV, the 15ADON chemotype was the most frequently detected, while F. asiaticum merely contained 3ADON chemotype. In addition, the average diseased ear areas of 15ADON, NIV and 3ADON type strains were 17.87%, 17.20%, and 12.37%, respectively. 【Conclusion】There existed frequent gene exchanges between different FGSC geographical populations, especially in adjacent regions. 15ADON type is the predominant toxigenic chemotype of FGSC in 7 geographic regions. The pathogenicity of F. graminearum is the strongest, followed by F. asiaticum, F. boothii, and F. meridionale according to the percentage of the diseased area caused by different Fusarium species. In this study, the correlation between toxigenic chemotypes and Fusarium species is not significant, and the pathogenicity is mainly related to Fusarium species.

The Evolutionary Dynamics and Adaptive Evolution of Tomato Chlorosis Virus

ZOU LinFeng,TU LiQin,SHEN JianGuo,DU ZhenGuo,CAI Wei,JI YingHua,GAO FangLuan

Scientia Agricultura Sinica. 2020, 53(23): 4791-4801. doi:10.3864/j.issn.0578-1752.2020.23.006

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【Objective】Tomato chlorosis virus (ToCV), a member of the genus Crinivirus in the family Closteroviridae, has emerged as one of the most prevalent viruses in tomato and other vegetable plants in China. The objectives of this study are to investigate the evolutionary history of ToCV, particularly when and where this virus was introduced into China, and to elucidate its adaptive evolutionary mechanism. 【Method】A specific primer pair flanking the coat protein gene (CP) of ToCV was designed. The CP of 13 randomly selected ToCV isolates from China was sequenced after reverse transcription and amplification using this primer pair. In addition to the novel sequence data, all published CP sequences with known sampling dates and geographic locations were obtained to assemble a total data set consisting of 103 CP sequences. A date-randomization test (DRT) was performed to assess the temporal signal in the data set and Bayesian phylodynamic framework was used to reconstruct the temporal dynamics of ToCV. Simultaneously, phylogeny-trait association analysis was used to identify whether geographic factors are involved in the adaptive evolution of ToCV. 【Result】A fragment with expected size (≈800 bp) was obtained from all the 13 ToCV-positive samples selected in this study. The CP of the 13 ToCV isolates shared more than 98% nucleotide identity with the reported sequences of other ToCV isolates. The 95% credibility intervals of rate estimate from the real sampling dates did not overlap with those from 10 replicate data sets with cluster permuted sampling date, indicating the presence of sufficient temporal signal in the sequence data allowing us to proceed with the Bayesian molecular dating analyses. Bayesian phylogenetic analyses showed that the estimated time of the most recent common ancestor of ToCV was 1920 CE (95% credibility interval 1849-1976) and the ToCV in mainland China was introduced from the USA at around 2005 CE. ToCV has been evolving at a rate of 1.12×10 ^-3 substitutions/site/year (95% credibility interval 6.08×10 ^-4-1.73×10 ^-3). This substitution rate is comparable to those estimated for animal RNA viruses, indicating that ToCV has been undergoing rapid evolutionary dynamics. In addition, significant association index, parsimony score and maximum monophyletic clade were observed when ToCV isolates were grouped by geographic origin (P<0.01), indicating a strong association between geographic factors and the adaptive evolution of ToCV. 【Conclusion】ToCV has been undergoing rapid evolutionary dynamics and its evolution was driven by geographic adaptation. These findings will be helpful in increasing our knowledge on the epidemiology of ToCV and have potential implications for developing more effective control strategies against this pathogen.

The Structure Characteristics and Biological Functions on Regulating Trehalose Metabolism of Two NlTret1s in Nilaparvata lugens

YU WeiDong,PAN BiYing,QIU LingYu,HUANG Zhen,ZHOU Tai,YE Lin,TANG Bin,WANG ShiGui

Scientia Agricultura Sinica. 2020, 53(23): 4802-4812. doi:10.3864/j.issn.0578-1752.2020.23.007

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【Objective】 Trehalose is the main blood sugar substance in insects and plays an important role in insect development and physiological activities. Among them, trehalose transporter (Tret) plays a key role in the transportation of trehalose from trehalose-producing tissues (such as fat body) to trehalose-consuming tissues. The objective of this study is to explore the biological functions of these two NlTret1s in brown planthopper (Nilaparvata lugens) by analyzing the sequence structure of two NlTret1s and further suppressing the expression of NlTret1s. 【Method】Taking these two NlTret1 sequences as the research object, the protein structure and homology with other insects were analyzed by bioinformatics technology. The RNA interference (RNAi) technology was used to inject the synthetic exogenous dsRNA (double-stranded RNA) into the laboratory feeding population of N. lugens, to inhibit the expression of the NlTret1s. The total RNA was extracted to synthesize the first-strand cDNA using reverse transcription Kit, qRT-PCR (quantitative real-time PCR) technology was used to detect the RNAi effect of dsNlTret1s and the expression of related genes in the trehalose metabolism pathway in N. lugens after RNAi, and finally glucose, trehalose and glycogen content as well as trehalase enzyme activity were determined. 【Result】 Bioinformatics analysis showed that the open reading frames of NlTret1-like X1 and NlTret1-2 X1 are 1 920 and 1 578 bp in length, encoding 639 and 525 amino acids, respectively. The predicted protein molecular weights are 69.29 and 58.71 kD, and the isoelectric points are 8.32 and 8.36, respectively. The secondary structure of NLTret1-like X1 and NLTret1-2 X1 is mainly composed of helix and coil. Conservative domain analysis showed that they all belong to the MFS family. The results of evolutionary analysis showed that Tret1 of different insects had high homology, and N. lugens was closely related to other hemiptera insects. Compared with the dsGFP group, the target gene was inhibited significantly after injection with dsNlTret1-like X1 or dsNlTret1-2 X1. Furthermore, the content of glycogen and glucose in N. lugens did not change significantly, but unlike the dsNlTret1-2 X1 group, the trehalose content of N. lugens was significantly increased with the injection of dsNlTret1-like X1. Meanwhile, the expression of the TPS1, TPS2, TRE1-1, TRE1-2 and TRE2 in N. lugens was significantly down-regulated after the NlTret1-like X1 knocked down for 48 h. The expression of TPS1, TPS2 and TRE1-1 in N. lugens also decreased significantly after injection with dsNlTret1-2 X1 for 48 h, while TRE1-2 and TRE2 showed a very significant upward trend. Moreover, after injection of dsNlTret1-like X1, the activities of soluble trehalase and membrane-bound trehalase were significantly reduced, but there was no significant change after injection with dsNlTret1-2 X1. 【Conclusion】These two Tret1s of N. lugens play different functions in different tissues, among which NlTret1-like X1 plays a more significant role in the specific transport of trehalose involved in energy supply. The results are helpful to explore the regulatory mechanism of Tret1 regulating the balance of trehalose metabolism in insects or invertebrates, and provide a theoretical basis for the future control of pests by regulating blood sugar balance, such as N. lugens.

Problems in Fertilization and Fertilizer Reduction in Wheat Production of China

WANG ZhaoHui

Scientia Agricultura Sinica. 2020, 53(23): 4813-4815. doi:10.3864/j.issn.0578-1752.2020.23.008

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Evaluation of Farmers’ Fertilizer Application and Fertilizer Reduction Potentials in Major Wheat Production Regions of China

HUANG QianNan,DANG HaiYan,HUANG TingMiao,HOU SaiBin,WANG ZhaoHui

Scientia Agricultura Sinica. 2020, 53(23): 4816-4834. doi:10.3864/j.issn.0578-1752.2020.23.009

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【Objective】It is of great significance to understand problems in farmers’ fertilizer application and the potential to reduce the fertilizer rates, with the purpose to realize scientific fertilizer recommendation and reasonable reduction of fertilizer input in major wheat production regions of China. 【Method】A three-year long in-farm fertilization survey and collection of soil and plant samples were carried out to analyze and evaluate the fertilization status and fertilizer reduction potentials based on the wheat yield levels, nutrient requirement and soil nutrient supply capacities in major wheat production regions of China. 【Result】The average wheat grain yields were significantly and linearly correlated with their biomass, with the average to be 6.0 and 13.2 t·hm ^-2 for farmers over all the major wheat regions in China, respectively. However, the yields showed no significant correlation with the fertilizer application rates and soil nutrients, and the average application rates for nitrogen (N), phosphorus (P₂O₅) and potassium (K₂O) were 191.1, 112.8 and 53.4 kg·hm ^-2, respectively. In the spring wheat region, the averages were correspondingly 171.7, 108.9 and 10.6 kg·hm ^-2, 154.3, 111.8 and 32.6 kg·hm ^-2 in dryland wheat region, 236.4, 128.1 and 74.0 kg·hm ^-2 in wheat-maize region, and 177.5, 77.0 and 71.8 kg·hm ^-2 in rice-wheat region, respectively. For the N, there were less farmers, only 34% over applied fertilizers in the spring wheat region, and then it was 42% in wheat-maize region, 55% in rice-wheat region, and 63% in dryland wheat region, with the low-yielding farmers to be the focus of N fertilizer reduction, the reduction potential to be 43.6%, and the average N reduction of 2.3-135.5 kg·hm ^-2. The problems for over P fertilizer application were more obvious, with 63%, 87%, 68% and 57% of farmers to apply excessive P fertilizer respectively in the four regions. Even at the high-yielding levels, there were still more than 50% of farmers apply excessive P fertilizer, and all the farmers need to reduce their P fertilizer, with the average reduction to be 3.8-91.1 kg P₂O₅·hm ^-2, and dryland wheat region of the largest reduction potential, which was 55.6% of their current P rates. Situation for K application was variable with regions. In spring wheat region, 84% of famers applied insufficient K fertilizers, with an average of 22.8 kg·hm ^-2 extra K₂O needed to be applied. While, in dryland wheat, wheat maize and rice wheat regions, 43.2%, 25.7% and 56.0% of their current K fertilizer application should be reduced, with the low yielding farmers to be the key in K fertilizer reduction and the average reduction of 31.7-45.9 kg K₂O·hm ^-2. 【Conclusion】Fertilizer application and its reduction potential were found to vary with the yields and regions for wheat farmers in China. Situation of excessive fertilization was more serious for low- and medium-yielding farmers. Application of N and K fertilizers should be reduced according to the wheat yield levels, and all farmers should pay special attention to their over P fertilizer application, and reduce the P application rates to reasonable levels. Fertilizer reduction potential was the highest in the dryland wheat region for N and P fertilizers, and in rice wheat region for K fertilizer.

Nutrient Resource Quantity from Main Grain Crop Straw Incorporation and Its Enlightenment on Chemical Fertilizer Reduction in Wheat Production in China

LI TingLiang,WANG YuFeng,WANG JiaHao,LI Li,XIE JunYu,LI LiNa,HUANG XiaoLei,XIE YingHe

Scientia Agricultura Sinica. 2020, 53(23): 4835-4854. doi:10.3864/j.issn.0578-1752.2020.23.010

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【Objective】The objective of this study was to determine the quantity and distribution of crop straws from rice, wheat and maize production, and the contained nutrient resources in the main grain crops planting regions of China, so as to provide a scientific basis for straw fertilizer utilization and reasonable reduction of chemical fertilizer in agricultural production in China. 【Method】 Based on data/information from National Bureau of Statistics of China and published literature, the amount of crop straws and the contained nutrient resources were estimated in rice, wheat and maize planting areas. The distribution of crop straws and the contained nutrient resources, the nutrient release of straw incorporation in next-stubble crops production, and chemical fertilizer proper reduction rate of wheat production were further analyzed in different agricultural regions of China. 【Result】The results showed that the ratio of straw to grain of rice, wheat and maize in China was 1.01, 1.14 and 1.25 by estimation of literature data, respectively. The annual yield of straw of the three major grain crops in China was 653.866 million tons during 2014-2018, among which rice, wheat and maize accounted for 32.3%, 22.7% and 45.0%, respectively. The crop straws were mainly produced in North China, Middle and Lower Reaches of the Yangtze River and Northeast China, accounting for 73.3% of the total national crop straw yields. The rice straw (50.7%) was mainly distributed in Middle and Lower Reaches of Yangtze River, the wheat straw (59.0%) was mainly distributed in Northeast China, and the maize straw were mainly distributed in Northeast China (33.7%) and North China (30.4%). A large number of literature data statistics showed that the nitrogen (N) average content of rice, wheat and maize straw was 0.78%, 0.64% and 0.85%, the phosphorus (P₂O₅) average content was 0.42%, 0.27% and 0.53%, the potassium (K₂O) average content was 2.31%, 1.53% and 1.59%, respectively, and the total nutrient content of straw (N+P₂O₅+K₂O) was expressed as rice>maize>wheat. The nutrient resources of three major grain crops straw were 5.098 million tons of N, 2.847 million tons of P₂O₅, and 11.83 million tons of K₂O. The distribution of total nutrient components in different agricultural areas was as follows: Middle and Lower Reaches of Yangtze River (26.0%)>North China (25.4%)>Northeast China (21.3%)>Northwest China (11.1%)>Southwest China (10.5%)>Southeast China (5.6%). The release rates of nitrogen from rice, wheat and maize straw returning to the field were 54.9%, 51.4% and 61.9%, that of phosphorus were 60.9%, 65.3% and 73.0%, and that of potassium were 90.1%, 93.3% and 92.3%, respectively, which showed as potassium>phosphorus>nitrogen. The annual amount of nutrient returned to field from three major grain crops straw (the amount of substitution of chemical fertilizer) contained 2.940 million tons of N, 1.941 million tons of P₂O₅and 10.839 million tons of K₂O, with a total amount of 15.72 million tons. Among them, the nutrient (N+P₂O₅+K₂O) release from maize straw return in the next crop growing period was the highest, accounting for 44.6% of the total amount of annual nutrient return. Straw incorporation had high potential of chemical fertilizer substitution for wheat production in China. In wheat monoculture area, the total wheat straw returning to the field could substitute chemical fertilizers input rate of 4.6 kg N·hm ^-2, 7.8 kg P₂O₅·hm ^-2and 65.3 kg K₂O·hm ^-2 theoretically. In the wheat-maize rotation area, the total amount of maize straw returned to the field could substitute chemical fertilizers input rate of 39.4 kg N·hm ^-2, 28.9 kg P₂O₅·hm ^-2and 109.9 kg K₂O·hm ^-2 in the wheat production season theoretically. In the rice-wheat rotation area, the total amount of rice straw returned to the field could substitute chemical fertilizers input rate of 29.9 kg N·hm ^-2, 17.8 kg P₂O₅·hm ^-2and 145.1 kg K₂O·hm ^-2 in the wheat production season theoretically. 【Conclusion】The annual yield of rice, wheat and maize straw in China was 211.415 million tons, 148.431 million tons and 294.020 million tons, respectively, with a total of 653.866 million tons. The straws of three major grain crops could provide 2.940 million tons of N, 1.941 million tons of P₂O₅ and 10.839 million tons of K₂O annually under straw returning. More than 70% of straw and nutrients resources were distributed in North China, Middle and Lower Reaches of the Yangtze River and Northeast China. In the wheat production area, the total amount of straw returned from the previous crop could substitute chemical fertilizers input rate of 4.6 to 39.4 kg N·hm ^-2, 7.8 to 28.9 kg P₂O₅·hm ^-2and 65.3 to145.1 kg K₂O·hm ^-2 in theory.

Potential Analysis of Organic Fertilizer Substitution for Chemical Fertilizer in Spring Wheat Regions of China

JIA LiGuo,SHI XiaoHua,SUYALA Qiqige,QIN YongLin,YU Jing,CHEN Yang,FAN MingShou

Scientia Agricultura Sinica. 2020, 53(23): 4855-4865. doi:10.3864/j.issn.0578-1752.2020.23.011

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【Objective】The objective of this study was to clarify the major problems of nutrition management in spring wheat production and to raise the suitable way on the organic fertilizer substitution for chemical fertilizer in different provinces and regions of China, so as to provide reference for Zero Growth Action of Chemical Fertilizers and sustainable development in spring wheat production regions. 【Method】A total of 2 166 survey data were collected from farmer interview and related literature retrieval, based on which the major problems were analyzed in different spring wheat regions of China. The data in 2018 of major livestock numbers, planting area and yield of spring wheat were downloaded from the National Bureau of Statistics of China. The nutritional contents of livestock manure, wheat straw and green manure plants were checked from published literatures, then the potential of organic fertilizer substitution for chemical fertilizer in different spring wheat regions was qualified. Comprehensive considered the climate, soils and the status of organic resources, the suitable ways of organic fertilizer substitution were suggested in different spring wheat regions. 【Result】Overuse of chemical nitrogen and phosphorus fertilizer but the deficiency of potassium and organic fertilizer dressing was the major problem of nutrition management in spring wheat. Especially, it was especially serious on nitrogen overuse in western Inner Mongolia and Ningxia irrigated spring wheat. The number of large livestock and sheep in spring wheat region was accounted for about 1/3 and 1/2 of that in China, respectively. The nutrient amount of N, P₂O₅ and K₂O from livestock manure was 238.6×10 ⁴, 57.6×10 ⁴and 141.0×10 ⁴t per year respectively, accordingly the potential of organic fertilizer from livestock manure substitution for chemical fertilizer was 78.2%, 48.1% and 43.1% for spring wheat, respectively. The total amount of straw returning to farmland in spring wheat region was 410.2×10 ⁴t per year. Available N, P₂O₅ and K₂O in straw was 11.4, 2.5 and 31.9 kg·hm ^-2 returning to farmland, accordingly in-season potential of organic fertilizer from wheat straw substitution for chemical fertilizer was 4.9%, 3.3% and 22.7%, respectively for spring wheat. Green manure plants returning to farmland was enough for meeting the nutrient requirement of spring wheat if the nutrients could release to soil completely. 【Conclusion】Based on the problems of overuse and unreasonable application of chemical fertilizer in spring wheat in China, three ways of organic fertilizer substitution including livestock manure, straw returning and green manure can be selected for resolving. By application of livestock manure the substitution potential for chemical fertilizer was 78.2%, 48.1% and 43.1%, respectively. The in-season substitution potential of organic fertilizer was 4.9%, 3.3% and 22.7%, respectively. Green manure, as organic fertilizer substitution, was sufficient for nutrient requirement of spring wheat if the nutrients could release to soil completely. The suitable way of organic fertilizer substitution should consider local resource, input and technology comprehensively.

Effects of Organic Fertilizers Replacing Chemical Fertilizers on Yield, Nutrient Use Efficiency, Economic and Environmental Benefits of Dryland Wheat

ZHANG QiRu,XIE YingHe,LI TingLiang,LIU Kai,JIANG LiWei,CAO Jing,SHAO JingLin

Scientia Agricultura Sinica. 2020, 53(23): 4866-4878. doi:10.3864/j.issn.0578-1752.2020.23.012

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【Objective】In order to provide the fertilization basis for scientific and efficient production of dryland wheat, the effects of organic fertilizer and biological organic fertilizer replacing chemical fertilizer on wheat yield, nutrient utilization, economy and soil environment were monitored continuously for 5 years. 【Method】From 2013 to 2018 in the dryland wheat field area of Hongtong County, Shanxi Province, there were four different fertilization patterns, including farmer pattern (FP), optimized fertilizers pattern (OF), optimized fertilizers+organic fertilizers pattern (OFM), and optimized fertilizers+biological organic fertilizers pattern (OFB), which were used to analyze the effects on the yield composition, economic benefits, nutrient absorption and transfer characteristics, fertilizer use efficiency, and soil environment of dry wheat in the Loess Plateau. 【Result】(1) Compared with the FP treatment, the average nitrogen fertilizer application was reduced by 35%, and the grain yield was significantly increased by 17.2%-21.4%, and net income was significantly increased by 44.3%-54.7% under OFM and OFB treatments, respectively; compared with the OF treatment, the average nitrogen substitution rate of OFM and OFB in five years was 40%, the spike number per hectare and 1000 grain weight were increased, and the grain yield was significantly increased by 6.0%-9.8%, and net income was significantly increased by 12.9%-21.0% under OFM and OFB treatments, respectively. (2) Compared with OF treatment, the contents of nitrogen in grain was significantly increased by 9.6%-12.8%, and the contents of phosphorus in grain was significantly increased by 12.5%-17.9% under OFM and OFB treatments, respectively; the transport amount at pre-anthesis of nitrogen, phosphorus and potassium of grain and the soil absorption after bloom were also increased, especially the soil absorption of nitrogen and phosphorus after anthesis was significantly increased by 48.8%-50.5% and 70.5%-76.2% under OFM and OFB treatments, respectively.(3) Compared with the OF treatment, the agronomic efficiency and partial productivity of potassium fertilizer under OFM treatment were significantly increased by 33.9% and 6.2%, respectively. The results showed that the apparent recovery rate of nitrogen and phosphorus fertilizer was increased by 48.6% and 65.5%, the agronomic efficiency of nitrogen and potassium fertilizer up by 71.3% and 51.3%, respectively, and the partial productivity was increased by 20.3% and 10.0%, respectively. (4)Organic fertilizers and biological organic fertilizers instead of chemical fertilizer increased soil fertility (organic matter, total nitrogen, available phosphorus, available potassium contents), nitrate nitrogen residue in soil surface was significantly decreased by 9.6%-23.0%, and there was no obvious leaching phenomenon of nitrate nitrogen in 2 m soil layer after 5 years. 【Conclusion】Instead of chemical fertilizer, organic fertilizers and biological organic fertilizers could improve the absorption of nitrogen, phosphorus and potassium in wheat grain, promote the absorption of nitrogen and phosphorus in soil after anthesis, improve the utilization efficiency of fertilizer, and significantly reduce the residue of nitrate nitrogen in soil, and alleviate soil alkalization, which was helpful to improve soil fertility and ultimately obtain higher economic and environmental benefits. Therefore, it was an important measure for the efficient and sustainable production of dryland wheat fields and the development of green agriculture.

Benefits of Yield, Environment and Economy from Substituting Fertilizer by Manure for Wheat Production of China

LI YongHua,WU XuePing,HE Gang,WANG ZhaoHui

Scientia Agricultura Sinica. 2020, 53(23): 4879-4890. doi:10.3864/j.issn.0578-1752.2020.23.013

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【Objective】 Substituting fertilizer by manure is a key measure to reduce the amount of chemical nitrogen (N) fertilizer while maintaining yield. However, the complex relationships among grain yield, environmental costs, and economic benefits of substituting fertilizer by manure were still not clear for wheat production. This study quantified the agronomic, environmental and economic benefits of substituting fertilizer by manure, aiming to provide a reference for the application of substituting fertilizer by manure for wheat production of China. 【Method】 In this study, the effects of substituting fertilizer by manure on the agronomic, environmental and economic benefits of wheat production in China were assessed through Meta-analysis. 【Result】Substituting fertilizer by manure did not reduce wheat yield under the condition that the use of chemical nitrogen fertilizer was reduced by an average of 43%. The replacement ratio of manure was less than 15% and 15%-30%, and the yield significantly increased by 8% and 5%, respectively; while the replacement ratio of manure was higher than 30%, the yield did not significantly increase. Net economic benefit of wheat production was influenced by the replacement ratio of manure and the type of substituted manure. The replacement ratio of manure was less than 30%, and the net economic benefit showed no significant diffidence; while the replacement ratio of manure was higher than 45%, net economic benefits decrease by 18%, 68% when using cow manure and commercial manure as substituting fertilizer. Substituting fertilizer by manure reduced NH₃ volatilization by 24% in whole wheat growing season, and decreased nitrate residue by 16% at wheat harvest stage, but increased N₂O emission by 32% in whole wheat growing season. Further analysis indicated that soil properties, manure types, climatic conditions, test duration, yield and fertilization level could regulate wheat yield of substituted manure. Compared with application of chemical fertilizer treatment, the grain yield in substituting fertilizer by manure increased by 4% when soil organic matter>15 g·kg ^-1, while there was no difference when soil organic matter<15 g·kg ^-1. Yield in substituting fertilizer by manure increased by 8% when soil pH<7, while there was no difference when soil pH>7. When chicken manure, pig manure and commercial organic manure was used as substituted manure, grain yield increased by 6%, 6% and 4%, respectively, while there was no difference when cow manure was considered as substituted manure. Substituting fertilizer by manure treatment significantly increased yield by 4% in the areas with annual precipitation >600 mm, but there was no difference in the areas with annual precipitation ≤600 mm. Substituting fertilizer by manure increased yield by 5% in areas with average annual air temperature>15℃, but there was no difference in the areas with average annual air temperature <15℃. The duration of the experiment, the different yield and fertilization levels could affect wheat yield of substituted manure. Compared with the application of chemical fertilizer treatment, the grain yield in substituting fertilizer by manure increased by 6% when duration of the experiment more than a decade, while there was no difference when duration of the experiment less than a decade. Yield in substituting fertilizer by manure increased by 4% when under the condition of high nitrogen application rate, while there was no difference when low nitrogen application rate. The substitution of manure at low yields significantly increased wheat yield by 9%, while the substitution of manure at medium and high yields no difference. 【Conclusion】 Substituting fertilizer by manure did not reduce wheat yield under the condition that the use of chemical nitrogen fertilizer was reduced by an average of 43%. Substituting fertilizer by manure had the potential to substantially reduce the amount of chemical N fertilizer and N loss while maintaining wheat yield, but did not increase economic income. It was very important for increasing wheat yield, protecting ecological environment and increasing economic income to adopt suitable manure types and ratio of substituting fertilizer by manure. The use of reasonable amount of chemical N fertilizer substitution and manure types had the potential to increase the yield of substituting fertilizer by manure in the areas of sufficient precipitation and high air temperature.

Requirement of Nitrogen, Phosphorus and Potassium and Potential of Reducing Fertilizer Application of Spring Wheat in Yellow River Irrigation Area of Ningxia

WANG XiNa,YU JinMing,TAN JunLi,ZHANG JiaQun,WEI ZhaoQing,WANG ZhaoHui

Scientia Agricultura Sinica. 2020, 53(23): 4891-4903. doi:10.3864/j.issn.0578-1752.2020.23.014

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【Objective】The aim of this study was to clarify fertilization and requirement of nitrogen (N), phosphorus (P) and potassium (K) in spring wheat production in Yellow River Irrigation Area of Ningxia (NYRIA), so as to provide a theoretical guide for rational fertilizer reduction and application. 【Method】Based on the investigation of farmers’ fertilizer application and field fertilization experiments, farmers’ yield levels and N, P, K fertilizer application of spring wheat were analyzed, and the grain yield, yield components, and N, P, K nutrient requirement were investigated. 【Result】The average spring wheat yield of farmers was (6 985±867) kg·hm ^-2 in NYRIA, and the percentage of high yield farmers was 82.7%. With the increase of yield, the excessive amount of N and P was decreased, and the application rates of K was insufficient. On average, the application rates of N, P and K were 294, 162 and 49 kg·hm ^-2, respectively, with 97.1% of farmers over applied N by 69-114 kg·hm ^-2, and 20.5% of farmers over applied P by 18-42 kg·hm ^-2, while K inputs were generally insufficient with an amount of 30-51 kg·hm ^-2. The N fertilization experiment showed that shoot biomass, grain yield, harvest index and grain number per ear all increased significantly at N rates of 120-240 kg·hm ^-2, and all reached the highest at the application of N 180 kg·hm ^-2. At the same time, the amount of N, P, and K absorbed by the grain also reached to the maximum value of 168.2, 23.9, and 23.2 kg·hm ^-2, respectively. Nitrogen application showed no significant effect on N harvest index, the average of which was 56.5%, and tended to increase P harvest index but decrease K harvest index. Nitrogen requirement reached to 45.8 kg·Mg ^-1 at N rate of 180 kg·hm ^-2, being 19.6% higher than that of no N application. P requirement decreased from 6.0 kg·Mg ^-1 at no N application to 5.3 kg·Mg ^-1 at N rate of 240 kg·hm ^-2, while K requirement increased from 42.6 to 49.7 kg·Mg ^-1. The P experiment showed that shoot biomass and grain number per ear decreased significantly with the increase of P rates, while 1 000 grain weight and harvest index increased significantly, so there was no significant difference in grain yield over P rates. Moreover, P application improved N uptake and harvest index, being 28.6% and 27.9% higher respectively than that of no P application. Also, P fertilizer could promote P to transfer to grain because that P uptake and P harvest index were increased by 15.9% and 15.2%, respectively. However, it showed no significant effect on N and P requirement, but decreased K requirement from 68.1 kg·Mg ^-1 at no P application to 49.7 kg·Mg ^-1 at P₂O₅ rate of 120 kg·hm ^-2. The K experiment showed that the application of K had no significant effects on biomass, grain yield, harvest index, ears per hectare, and the content of N, P and K in grain. While the high K fertilizer application of 75 kg·hm ^-2 significantly reduced the number of grains per ear, but increased 1 000 grain weight and harvest index of N, P and K. Grain number per ear decreased by 9.1%, 1 000 grain weight increased by 7.6%, and harvest indexes of N, P and K were 57.2%, 73.5% and 7.3%, respectively. When applying 60 kg·hm ^-2 of K₂O, the demand for N, P and K reached the highest, which was 55.3, 5.5 and 57.6 kg·Mg ^-1, respectively, while the demand for N and K were both significantly reduced by 20.6% and 13.7% at K₂O rate of 75 kg·hm ^-2. It was concluded that proper application of K could increase the demand of N and K, while over application of K could reduce the demand of N and K. 【Conclusion】Reducing N fertilizer, regulating P fertilizer and properly adding K fertilizer input were still the key for spring wheat fertilization in NYRIA. The N requirement of spring wheat was ranged 38.3-57.2 kg·Mg ^-1, and could increase by applying the moderate amount of N, P and K fertilizer. The demand for P of spring wheat was 5.1-6.0 kg·Mg ^-1, which tends to increase with N rates and is not influenced by P and K amount. The K requirement of spring wheat was within 42.6-68.1 kg·Mg ^-1, which seems to increase with N rates and decrease with P rates, and also is reduced by high amount of K application. The suitable recommended application of N fertilizer was 120-180 kg·hm ^-2, which was 25%-60% lower compared with N application of farmers. Application of P fertilizer at 48-96 kg·hm ^-2 was more conducive to stabilize the yield of spring wheat and promote the transfer of N and P to the grain, which was reduced by 40.7%-70.3% compared with the average application of farmers. When the application of K fertilizer was 0-30 kg·hm ^-2, it was more benefit to stabilize the yield and increase the grain quality.

Light Regulation of Anthocyanin Biosynthesis in Horticultural Crops

WANG Feng,WANG XiuJie,ZHAO ShengNan,YAN JiaRong,BU Xin,ZHANG Ying,LIU YuFeng,XU Tao,QI MingFang,QI HongYan,LI TianLai

Scientia Agricultura Sinica. 2020, 53(23): 4904-4917. doi:10.3864/j.issn.0578-1752.2020.23.015

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Anthocyanins are among the most important flavonoid compounds in plants, which play significant roles in color formation of plant organ, such as flower and fruits, as well as antioxidant process. Light is one of the most important environmental factors affecting anthocyanin biosynthesis pathway, but it still remains unclear in the mechanism and signaling networks of light regulation of anthocyanin. This review briefly introduced the anthocyanin biosynthesis and transportation pathway, and summarized the molecular mechanism of anthocyanin transcriptional regulation by three kinds of transcription factors, including MYB, bHLH and WDR. In addition, it emphasized on the light signaling regulation of anthocyanin biosynthesis. The researches showed that the light environment (light intensity, light quality, and light duration) regulated the biosynthetic process of anthocyanin mainly through different light receptors (UVR8, CRYs, PHOTs, and PHYs), which affected the ubiquitination ability of COP1, the stability of HY5, and the stability of other light signal transcription factors, such as the phytochrome-interacting factors (PIFs). On the one hand, these light signal factors directly could bind to the promoters of MYB, bHLH and WDR, activate or inhibit these genes expression and then regulate the synthesis of anthocyanin. On the other hand, these light signal factors interacted with proteins of MYB, bHLH and WDR, affecting the stability of the MBW complex formed by them. In addition, these light signaling factors could also regulate anthocyanin synthesis through MBW independent pathways, such as HY5 also affect anthocyanin biosynthesis by regulating miR858. In addition, some unknown light signaling factors might directly or indirectly regulate anthocyanin synthesis genes and interacting with some vacuolar membranes proteins in a MBW independent manner, to change vacuolar acidification and regulate anthocyanin synthesis. At the same time, light signaling factors also affected some factors in the photosynthetic electron transport chain through MBW dependent or MBW independent pathways, then affected anthocyanin synthesis in plants. How these pathways were coordinated and which pathway was preferentially responded by light environments (light intensity, light quality, light duration)? This paper provided a basis to further investigate the molecular mechanism regulating anthocyanin biosynthesis by light signalings. The study explored the effective ways and target molecules for light regulation of anthocyanin accumulation, and created opportunities for the development of anthocyanin-rich horticultural crops through genetic and metabolic engineering, and light environmental management.

The Effects of Different Oxygen Concentration on Postharvest Physiology and Storage Quality of Yali Pear

DU YanMin,WANG WenHui,JIA XiaoHui,TONG Wei,WANG Yang,ZHANG XinNan

Scientia Agricultura Sinica. 2020, 53(23): 4918-4928. doi:10.3864/j.issn.0578-1752.2020.23.016

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【Objective】The aim of this study was to clarify the effects of environment oxygen concentration on postharvest physiology and storage quality, affording the theory basis to prevent the postharvest disorders and prolong the storage period of Yali fruit. 【Method】Pear samples were harvested from the commercial orchard in Shijiazhuang city, Hebei province. The fruits of uniform size and ripeness without insect pests or mechanical injury were selected as research materials. The selected fruits were put into controlled atmosphere chambers, and precooling at 8℃ for 48 h, and then declined by 1℃ for every 2 days, and finally storage at (0±0.5)℃ (RH>95%). Then the controlled atmosphere (CA) was established under different O₂ concentrations (1.0%, 2.0%, 3.0%, 5.0% and 10.0%) plus 0.5% CO₂, and taking air as control. Fruit firmness (FF), soluble solids content (SSC), titratable acidity (TA), ascorbic acid (AA), ethanol, respiration rate, ethylene production and the relative expression of ethylene biosynthesis genes PbACS and PbACO were determined after the fruits storage for 150 d, 210 d and 270 d, respectively. 【Result】The heart browning incidence and superficial scald incidence were both declined significantly under CA treatments during long period storage. More specifically, after storage for 270 d and shelf-life for 7 d, the disorder prevention efficiency were followed by the order of CK>10.0% O₂>5.0% O₂>1.0% O₂>3.0% O₂>2.0% O₂. Besides that, the lower O₂ treatments maintained TA and AA contents at a higher level in the early storage period, and delayed the yellow-turning of peel; ethanol was accumulated much more under the lower O₂concentration, which ranged from 150.89 mg?L ^-1 to 806.12 mg·L ^-1 after storage for 150 d; while with the increasing senescence browning of fruit in air, the ethanol content peaking at 1 500 mg·L ^-1 after storage for 270 d. 5.0% and lower O₂ inhibited the ethylene biosynthesis and delayed the peaking time, and 1.0%-3.0% O₂ could prohibit the relative expression of PbACO1, PbACO3 and PbACS1 genes in pericarp tissue significantly. 【Conclusion】1.0%—3.0% O₂ prohibited the superficial scald occurrence significantly, and delayed the decrease of TA and AA, inhibited the biosynthesis of ethylene and delayed the peaking time, down-regulated the relative expression of PbACO1, PbACO3 and PbACS1 genes in pericarp tissue, prolonged the storage period of Yali pear significantly. However, 1.0% oxygen treatment increased the risk of heart browning to some extent. Therefore, the optimum oxygen concentration was proposed at 3.0%-5.0% in commercial practice.

Regulation Mechanism of Carvone on Seed Potato Sprouting

GE Xia,XU Rui,LI Mei,TIAN JiaChun,LI ShouQiang,CHENG JianXin,TIAN ShiLong

Scientia Agricultura Sinica. 2020, 53(23): 4929-4939. doi:10.3864/j.issn.0578-1752.2020.23.017

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【Objective】 Carvone is a common monoterpene in plant essential oils. In this study, the partial regulation mechanism of carvone on seed potato sprouting was explored from the aspects of hormone regulation and membrane lipid peroxidation. 【Method】The cultivar Qingshu 9 was used as experimental material. The method of treatment was using 0.3 mL carvone per kilogram of tuber for 18 weeks, and then stopping chemical for 6 weeks before planting. At the same time, the treatment without using carvone was as control group. Sprout length, weight loss, abscisic acid (ABA), indoleacetic acid (IAA), gibberellic acid (GA₃), malondialdehyde (MDA) and proline (PRO) contents, superoxide dismutase (SOD), catalase (CAT), polyphenol oxidase (PPO) and peroxidase (POD) enzyme activities were determined under different storage periods (at 0, 6, 18, 20 and 24 week). Meanwhile, the bud meristem of seed potato was sectioned and analyzed by microimaging regularly. 【Result】 Carvone treatment could regulate potato sprouting and the sprout length, and reduce the weight loss of seed potatoes. After 24 weeks of storage, the sprout length of the treated seed potatoes was 4.77 mm, significantly lower than that of the control group (51.02 mm), and the weight loss was reduced by 49.76%. Carvone treatment inhibited the growth of apical buds by damaging apical meristem and vascular tissue, which did not affect the emergence of axillary buds. During storage, carvone treatment significantly increased the ABA content, inhibited the IAA content, delayed the peak of GA₃ content and the appearance of (GA₃+IAA) /ABA >1 inflection point. For the carvone reated potatoes, MDA content in potato was 15.04%-25.90% lower than control group; PRO content was 24.59%-54.45% higher than control group; the activities of SOD, CAT and PPO were 18.86%-35.56%, 14.34%-31.97% and 26.00%-36.41% higher than control group, respectively; POD activity of treated potatoes was 1.87-3.07 times that of control group. 【Conclusion】 Carvone treatment adjusted the ABA, IAA and GA₃ contents of seed potatoes by promoting/inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the mevalonate pathway, and regulated the inhibition of the apical bud. In addition, it increased antioxidant enzymes activities, reduced the degree of membrane lipid peroxidation and alleviated seed potato aging.

Effect of KISS1 Interference on the Function of Porcine Granulosa Cells in Porcine Ovary

LU SiYu,HE YingTing,ZHOU XiaoFeng,XIN XiaoPing,ZHANG AiLing,YUAN XiaoLong,ZHANG Zhe,LI JiaQi

Scientia Agricultura Sinica. 2020, 53(23): 4940-4949. doi:10.3864/j.issn.0578-1752.2020.23.018

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【Background】 As the basic structural and functional unit of ovary, the main functions of follicles are ovulation and hormone secretion. Granulosa cells proliferation can promote the development of follicles, while the excessive apoptosis of granulosa cells will inhibit the development of follicles, induce follicular atresia, reduce the estrus frequency and affect the reproductive capacity of female animals. Recent studies have found that the KISS1 plays an important role in ovary. 【Objective】 This study was aimed to explore the effects of KISS1 on apoptosis, cell cycle and estrogen secretion of porcine granulosa cells, through interfering the expression of KISS1, so as to provide some useful information in molecular regulation mechanism of KISS1 on porcine granulosa cells. 【Method】 To investigate the effect of knockdown KISS1 on the transcription of key genes on phosphatidylinositol 3-kinase (PI3K) signaling pathway in porcine granulosa cells, KISS1-siRNA were designed and transfected to porcine granulosa cells in vitro, and then quantitative real time PCR (qRT PCR) was applied to detect the transcription of KISS1. Then, flow cytometry, Annexin V-FITC, and ELISA were utilized to explore the effects of KISS1-siRNA on granulosa cell cycle, apoptosis, and secretion of E₂, respectively. Finally, qRT-PCR was applied to explore the effects of KISS1-siRNA on estrogen receptor and estrogen signaling pathway. 【Result】 In porcine granulosa cells, interfering KISS1 gene could significantly decrease the transcription of AKT1(P<0.05), while PIK3CG, PI3CI and PDK1 decreased with no significant difference (P>0.05). The mRNA levels of the genes, involving in activation of PI3K signaling pathway like FOXO3, TSC2, and BAD, were also decreased with no significant difference (P>0.05). Transfecting KISS1-siRNA to porcine granulosa cells could significantly promote cell apoptosis (P<0.01), arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E₂ secretion (P<0.01). After knockdown KISS1, the transcription of estrogen receptor ESR1 and ESR2, as well as Star, CYP17, 3B-HSD, 17B-HSD and CYP19A of estrogen pathway were also decreased significantly (P<0.05). 【Conclusion】 This study confirmed that KISS1 gene could participate in PI3K and estrogen signaling pathways. Interfering KISS1 could promote porcine granulosa cells apoptosis, arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E₂ secretion. It suggested that KISS1 played an important role in the division, growth and estrogen secretion of porcine granulosa cells.

Effects of Dietary Small Peptides on Production Performance and Expression of PepT1 mRNA in Digestive Tract of Fattening Yaks

MIAO JianJun,PENG ZhongLi,GAO YanHua,BAI Xue,XIE XinTing

Scientia Agricultura Sinica. 2020, 53(23): 4950-4960. doi:10.3864/j.issn.0578-1752.2020.23.019

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【Objective】This study investigated the effects of dietary small peptides on production performance, apparent digestibility of nutrients, blood indexes and expression of PepT1 mRNA in digestive tract of fattening yaks. 【Method】Health Maiwa male yaks (n=36, (180.98±20.57) kg body weight) were divided into 4 groups in a randomized complete block design, and each group had 9 male yaks. And they were fed 4 types of total mixed ratio diet with small peptides additions of 0, 0.75%, 1.50% and 2.25%, respectively. There was a pre-experimental period of 30 days, followed by a trial period of 80 days. Every yak’s weight was recorded before and after the trial begins, and the feed intake was also recorded every day. During the last week of the trial period, the fecal grab samples were collected for 3 consecutive days from each yak for routine analysis of nutrients. And five yaks were selected from each group to collect jugular vein blood and prepare serum to determine serum biochemical and immune indicators. At the end of the trial period, 5 yaks with blood collected were slaughtered, and the rumen, reticulum, omasum, abomasum, duodenum, jejunum, and ileum tissue were collected to determine the expression of PepT1 mRNA by using real time quantitative PCR. 【Result】(1) With the level of small peptides increasing, the dry matter intake, average daily gain and the feed gain ratio showed quadratic variation (P<0.05). The group with the best production performance was the 2.25% group. Compared with the control group and the 0.75% group, the dry matter intake and average daily gain were significant improved in the 2.25% group, but the feed gain ratio was significant decreased in the 2.25% group. (2) With increasing of small peptides levels, the degradation rate of neutral detergent fibre and acid detergent fibre linearly increased (P<0.01); the degradation rate of organic matter and crude protein presented quadratic variation (P<0.05). The 2.25% group had the highest digestibility. No significant difference existed between the degradation rate of calcium and phosphorus in four groups (P>0.05). (3) Across the range of small peptides supplementation levels, the content of alanine aminotransferase linearly decreased (P<0.05); the content of urea nitrogen linearly increased (P<0.05); the total protein showed linearly increased trend (P<0.10), and the content of aspartate aminotransferase showed quadratic variation (P<0.05). There were no significant effects on the content of alkaline phosphatase, glucose, cholesterol, triglyceride and immunoglobulins (IgG, IgA and IgM) in yak serum by adding different levels of small peptides. (4) The expression of PepT1 mRNA in the digestive tract of yaks from the highest to the lowest was the jejunum, ileum, duodenum, reticulum, omasum, rumen, and abomasum, while the expression of PepT1 mRNA in jejunum was significantly higher than that of the other gastrointestinal tracts (P<0.05). The expression of PepT1 mRNA in abdomen was significantly lower than those of jejunum and ileum. No significant difference was found between the expression of PepT1 mRNA in ileum, duodenum, reticulum, omasum, and rumen. With increasing of small peptides levels from 0 to 2.25%, the expression of PepT1 mRNA in rumen, reticulum and jejunum linearly increased (P<0.05), and small peptides levels had no significant effect on the expression of PepT1 mRNA in omasum, abomasum, duodenum and ileum (P>0.05). 【Conclusion】 In conclusion, dietary small peptides could improve the production performance, apparent digestibility of nutrients, liver function of fattening yaks, and promote the transport and absorption of small peptides in the digestive tract. In this research, the appropriate level of dietary small peptides of yaks was 2.25%.

Production of Citrus Triploids Based on Interploidy Crossing with Bendizao and Man Tangerines as Female Parents

XIE KaiDong,PENG Jun,YUAN DongYa,QIANG RuiRui,XIE ShanPeng,ZHOU Rui,XIA QiangMing,WU XiaoMeng,KE FuZhi,LIU GaoPing,GROSSER Jude W,GUO WenWu

Scientia Agricultura Sinica. 2020, 53(23): 4961-4968. doi:10.3864/j.issn.0578-1752.2020.23.020

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【Objective】The aim of this study was to create the citrus triploids based on diploid and tetraploid ploidy cross strategy. 【Method】The artificial pollination was conducted with diploid as female parent and tetraploid as male parent. Young fruits were sampled at 85 d after pollination (DAP) and immature seeds were extracted and subjected to in vitro embryo rescue. Following plantlet regeneration from the embryos, their ploidy level was determined by flow cytometry and root-tip chromosome counting, as well as the genetic origin determined using simple sequence repeat (SSR) markers. 【Result】A total of nine interploidy crosses were carried out by using Bendizao tangerine and Man tangerine as female parents and eight allotetraploid somatic hybrids and one doubled diploids as male parents. From all crosses, 2 749 flowers were pollinated and 489 fruits were set, with an average fruit setting rate of 17.8%. By conducting in vitro immature embryo rescue, totally 260 plants were regenerated from 2 239 seeds cultured. By determining their ploidy level using flow cytometry and root-tip chromosome counting, 141 seedlings were proven to be triploids. SSR analysis showed that all of 50 randomly selected triploid plants from Man tangerine × NS were the hybrids of their both parents. After one year growing in greenhouse, all triploids were grafted onto Poncirus trifoliata in the field to accelerate flowering. 【Conclusion】These triploid citrus plants obtained herein provided elite materials for potential seedless variety selection.

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