Table of Content

16 July 2023, Volume 56 Issue 14

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Population Genetic Analysis of Puccinia striiformis tritici in Main Winter-Increasing Areas Based on Virulent Phenotypes and Genotypes

GAO XinPei, ZHAO Jun, LIU BoFan, GUO Yi, KANG ZhenSheng, ZHAN GangMing

Scientia Agricultura Sinica. 2023, 56(14):  2629-2642.  doi:10.3864/j.issn.0578-1752.2023.14.001

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【Objective】To clarify the virulence structure and genetic diversity of Pst populations in the major winter-increasing areas of China, and to provide reference for the prevention and control of Pst and the rational layout of wheat resistance genes in the winter-increasing areas and the wheat production in Huang-huai-hai. 【Method】A total of 148 Pst isolates were collected and isolated from the major winter-increasing areas such as Sichuan Basin, Hubei and southern Henan, and the virulence phenotype was identified by using Chinese differentials and single-gene lines, and 17 pairs of KASP-SNP primers were used to mark the isolates and complete the genotype analysis. 【Result】Based on the Chinese differentials, 14 known races and 63 unknown pathotypes were identified, among which CYR34 (16.2%), G22-14 (12.2%), CYR32 (6.8%), CYR33 (5.4%) were the dominant races (pathotypes); based on the single-gene lines, 113 races (pathotypes) were identified, among which race1 (7.4%), race2 (3.4%), race3 (3.4%) were the dominant races (pathotypes). The Guinong 22 group was the largest epidemic group of Pst population in China’s winter-increasing area, and all tested Pst isolates did not infect single-gene lines varieties carrying Yr5 and Yr15. The virulence phenotype and genotype of CYR34 and G22-14 showed diversification by single-gene lines virulence identification and molecular marker, indicating that there was high differentiation within these two dominant races. The clustering based on the virulence data of two sets of differentials showed that the Pst populations in Sichuan Basin and southern Hubei were similar, while the Pst populations in northwestern Hubei and southern Henan were similar; the genetic clustering based on KASP-SNP molecular data showed that there was genotype differentiation between the Pst populations in Sichuan Basin, southern Hubei and northwestern Hubei, southern Henan; Structure analysis showed that Sichuan Basin, southern Hubei population mainly had two genetic backgrounds, northwestern Hubei, southern Henan population mainly had one genetic background; population genetic differentiation analysis showed that Sichuan Basin Pst population and southern Henan Pst population had the largest Fst value, which was 0.118, with the largest genetic difference and obvious genetic differentiation; northwestern Hubei population and southern Henan population had the smallest degree of genetic differentiation, Fst value was 0.010; gene flow analysis obtained Nm value between northwestern Hubei population and southern Henan population was 25.236, Nm>4, there was a high-level gene flow between them, northwestern Hubei and southern Henan population and Sichuan Basin population had Nm values of 2.923 and 1.864 respectively, both had a low-level gene flow; genetic diversity analysis results showed that Sichuan Basin, southern Hubei region Pst population had a high-level of genetic diversity, northwestern Hubei, southern Henan Pst population had a low-level of genetic diversity. The above conclusions all support that Sichuan Basin, southern Hubei population has genetic differentiation with northwestern Hubei, southern Henan population. 【Conclusion】Single-gene lines can accurately identify Chinese Pst races; Pst populations in China’s major winter-increasing areas have different sources.

Pigment Identification and Gene Mapping in Red Seed Coat of Soybean

CAO Jie, GU YongZhe, HONG HuiLong, WU HaiTao, ZHANG Xia, SUN JianQiang, BAO LiGao, QIU LiJuan

Scientia Agricultura Sinica. 2023, 56(14):  2643-2659.  doi:10.3864/j.issn.0578-1752.2023.14.002

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【Objective】To identify the key genes controlling anthocyanin synthesis and accumulation, to uncover changes in anthocyanin content of the seed coat during seed development, and the primary anthocyanin components responsible for the red seed coat of Taixingaijiaohong (TXAJH); and to lay the groundwork for a thorough understanding of the regulatory mechanism of red seed coat formation.【Method】Using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS), the anthocyanin composition and concentration of the yellow seed coat of soybean Suinong 14 (SN14) and the red seed coat of soybean TXAJH at various developmental stages were identified. The potential areas of red testa-related genes were first identified using bulked segregant analysis (BSA) on the recombinant inbred lines (RILs) made by crossing SN14 and TXAJH. Based on this discovery, we performed marker linkage analysis to restrict the candidate intervals and predict the candidate genes, and qRT-PCR to confirm the expression of the anticipated candidate genes.【Result】When seed coats from the four developmental phases of SN14 and TXAJH were analyzed, a total of 12 anthocyanins were discovered. Cluster analysis of total anthocyanins revealed substantial changes in the seed coat's anthocyanin composition between TXAJH and SN14 as well as between TXAJH before and after color development. The anthocyanin content of the SN14 seed coat gradually decreased as the seed developed, whereas the TXAJH seed coat's content increased quickly and remained stable. After the development of the seed coat's color, the anthocyanin contents of SN14 and TXAJH showed highly significant differences, and at the mature stage, the TXAJH seed coat's anthocyanin content was more than 200 times that of SN14. The crimson coloring of the TXAJH seed coat was largely due to cyanidin-3-O-glucoside (Cy-3-glu), peonidin-3-O-glucoside (Pn-3-glu), and petunidin-3-O-glucoside (Pt-3-glu). The candidate interval for the red seed coat gene on chromosome 8 was discovered at 8.66 Mb by BSA-seq association analysis. 27 polymorphic markers were used in the marker linkage analysis, which produced 10 haplotypes and reduced the candidate interval to 702 kb. Nonsynonymous variations in 37 genes between the parents were found during this interval, these include the genes for encode the anthocyanin reductase 1 (Glyma.08g062000), the bHLH transcription factor (Glyma.08g061300 and Glyma.08g063900), and the MYB transcript factor (Glyma.08g059900). These genes may be involved in regulating the biosynthesis of anthocyanins, and anthocyanin reductase 1 can convert anthocyanins to proanthocyanidins (PA). The results of gene expression analysis revealed that candidate genes and genes related to the anthocyanin biosynthesis pathway had comparable expression patterns in SN14 and TXAJH, and both were expressed at lower levels in SN14 and at higher levels in TXAJH. It was discovered that there was a significant link between the principal constituents of seed coat anthocyanins and the level of candidate gene expression.【Conclusion】The anthocyanin makeup of SN14 and TXAJH's seed coats differed, and Cy-3-glu, Pn-3-glu, and Pt-3-glu may be to blame for the TXAJH's seed coat's red hue. According to predictions, Glyma.08g059900, Glyma.08g061300, Glyma.08g062000, and Glyma.08g063900 will likely be a candidate gene for the red seed coat, in which Glyma.08g059900, Glyma.08g061300, and Glyma.08g063900 may control a number of anthocyanin biosynthesis pathway genes.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Water Use Characteristics of Wheat Rotated After No Tillage Plastic Film Mulching Maize with Reduced Water and Nitrogen

CAO YongGang, XU LongLong, CHAI Qiang, HU FaLong, YIN Wen, FAN ZhiLong, WANG QiMing, ZHAO Cai

Scientia Agricultura Sinica. 2023, 56(14):  2660-2672.  doi:10.3864/j.issn.0578-1752.2023.14.003

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【Objective】In view of the severe water shortage of resource-based irrigation areas in the west of the river oasis and the large amount and low utilisation of mulch and water and nitrogen inputs in crop production, the effects of no-tillage and two-year use of mulch on the water utilisation characteristics of subsequent crops under water and nitrogen reduction conditions were explored to provide a practical basis for the construction of water and nitrogen reduction and efficient water utilisation technologies in the trial area.【Method】From 2019 to 2021, a three-factor split plot experiment was carried out, with two tillage methods of conventional tillage (CT) and no-tillage (NT), two irrigation levels of 20% reduced irrigation (I1, 1 920 m³·hm^-2) and conventional irrigation (I2, 2 400 m³·hm^-2), and three N levels of 40% reduced N application (N1, 135 kg·hm^-2), 20% reduced N application (N2, 180 kg·hm^-2) and conventional N application (N3, 225 kg·hm^-2), to study the evapotranspiration, evaporation and water use efficiency of different treatments.【Result】The one film for two years can optimize the soil water environment before wheat sowing and reduce the ineffective water consumption during wheat growth period. Compared to CT, NT was beneficial to increase soil water content and water storage before wheat sowing, which increased by 16.9%-23.0% and 14.5%-16.5%, respectively; It can reduce the evapotranspiration (ET), total evaporation (E) and proportion of evaporation to evapotranspiration (E/ET) of wheat in the whole growth period by 3.5%-4.2%, 19.0%-20.2% and 16.8%-19.3%, respectively. With the reduction of irrigation and N application, the three indicators of evapotranspiration, evaporation and E/ET decreased. Compared with I2, the three indicators decreased by 6.1%-6.4%, 10.8%-11.1% and 5.5%-6.0%, respectively; Compared with N3, N2 and N1 decreased by 2.2%-3.9%, 4.2%-10.9% and 1.7%-7.2%, respectively; Under the interaction of tillage and irrigation, evapotranspiration and E/ET of NTI1 decreased by 0.6%-6.6% and 17.4%-17.6% respectively compared with CTI1; Integrated water and nitrogen reduction for two years with no tillage and one film to further optimize water consumption structure, the three indicators for NTI1N2 were reduced by 11.0%-12.9%, 28.3%-47.6% and 22.5%-26.4% respectively compared to CTI2N3. Compared with CT, NT significantly improved wheat grain yield and water use efficiency by 4.8%-6.3% and 9.0%-9.5% respectively. Under NT and CT conditions, there was no significant difference in grain yield and water use efficiency (WUE) between I1N2 and I2N3, I2N2 wheat. Comparing wheat grain yield and water use efficiency under different treatments, NTI1N2 was the highest, increasing grain yield and WUE by 5.1%-6.6% and 5.7%-6.2%, respectively, compared to the control CTI2N3.【Conclusion】In the Hexi irrigation area, no-tillage and one-film two-year rotation of maize for wheat, irrigation of 1 920 m³· hm^-2 and N application of 180 kg· hm^-2 in the whole growth period, is a production technology suitable for water and nitrogen conservation, high wheat yield and efficient water use in this area.

Analysis on Key Production Techniques of Cotton with Good Quality and High Yield in Xinjiang

LOU ShanWei, TIAN LiWen, LUO HongHai, DU MingWei, LIN Tao, YANG Tao, ZHANG PengZhong

Scientia Agricultura Sinica. 2023, 56(14):  2673-2685.  doi:10.3864/j.issn.0578-1752.2023.14.004

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Although the cotton of Xinjiang has achieved high quality and high yield, its technological leadership and influence are still insufficient. To figure out the key technologies of high-quality cotton production in Xinjiang which can reflect the technical characteristics of cotton production comprehensively and objectively so that other areas where cotton is grown can understand cotton production technology in Xinjiang and show level of cotton production to the world. In addition, this article is to summarize experience, strengthen the popularization and further maturing of advanced technology, improve the application efficiency, and promote the promotion of cotton production technology in Xinjiang to other cotton regions. This paper based on years of experimental data and research results, awards and patented achievements such as “Innovation and application of light and efficient key technologies in cotton industrialization in Xinjiang” “Seeding Protection Method in Cotton Precision Seeding Field in Xinjiang”, combined with literature review, academic exchanges, visits and research. This analysis is carried out according to the order and tightness with technology corresponding to the cotton growth process. A series of key technologies are summarized, including: the seminal seeding and the supporting seedling technology, cotton field short-term, dense, early group control technology, water and fertilizer integration technology, the whole process of tube harvest mechanization and its supporting technology, pest and disease control technology. The relatively perfect cotton production technology system in Xinjiang has been constructed through improvement, optimization, maturation, innovation and application of key technologies, so that the technical level of cotton production in Xinjiang has been greatly improved to cover less than 9% of the world's planted cotton area, harvest more than 20% of the world's cotton, and lead the world in terms of single production level, quality in the world, and the total production, single production, and commodity transfers have been maintained for 28 consecutive years for the first time in the country. The in-depth analysis of innovation, optimization and practicability of cotton production technology in Xinjiang can help cotton regions better understand the inevitability and advanced of high yield and quality of cotton in Xinjiang. Practice also proves that, with the support of a series of key technologies, cotton in Xinjiang has achieved high-quality production and has become the region with the highest per unit yield and mechanization of cotton in China, which means that its technology can guide production practice and promote application.

Spatial-Temporal Variations of High Temperature During Flowering Period in Maize-Producing Areas of China Under Climate Change

FU ZhenZhen, ZHU GuangXin, LIU ZhiJuan, GUO ShiBo, LI E, YANG XiaoGuang

Scientia Agricultura Sinica. 2023, 56(14):  2686-2700.  doi:10.3864/j.issn.0578-1752.2023.014.005

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【Objective】Under the background of global warming, the frequent occurrence of extreme high temperature would threaten crop production greatly. Therefore, the spatial-temporal variations of high temperature during crop growth period based on several heat stress index were cleared, which was crucial for developing effective disaster risk management and adaptation measures. 【Method】In this study, the maize potential planting area was focused on. Based on the daily maximum temperature data from 1981 to 2060 in two Shared Socioeconomic Pathways scenarios (SSP1-2.6 and SSP5-8.5) of Coupled Model Intercomparison Project Phase 6 (CMIP6) and maize phenology data, we analyzed the spatial distribution and temporal trend of the heat stress intensity (HSI), accumulated heat stress days (AHSD), and heat degree-days (HDD) during flowering period of maize in the baseline period (1981-2014) and future period (2015-2060).【Result】From 1981 to 2014, the HSI, AHSD and HDD during the flowering period of maize were the largest in Huang-Huai-Hai (HHH) and Northwest China (NWC), with the average value of 32.3 and 33.8 ℃, 8.4 and 9.8 d, 22.9 and 40.3 ℃·d, respectively. Due to climate warming, the high temperature during the flowering period of maize in China was characterized by long duration and wide range under the two climate scenarios, especially in SSP5-8.5. The largest temporal trend of HSI and AHSD occurred in Northern China (NC), under SSP1-2.6 and SSP5-8.5. The increasing trend of HSI were 0.97 and 1.16 ℃·(10a)^-1, and the increasing trend of AHSD were 0.73 and 1.11 d·(10a)^-1. The largest temporal trend of HDD occurred in HHH, under SSP1-2.6 and SSP5-8.5, with the increasing trend of 2.68 and 5.26 ℃·d·(10a)^-1. 【Conclusion】In the future, the high temperature during the flowering period of maize in China was characterized by long duration and wide range, especially for HHH and NC. The former was mainly due to the high base temperature, and the latter was due to the large warming trend. The loss caused by high temperature could be reduced by selecting high temperature resistant varieties, adjusting the sowing window, adopting water, fertilizer, and chemical management measures.

PLANT PROTECTION

Overexpressing NPR1 from Arabidopsis thaliana Enhanced Resistance to Fusarium Wilt and Powdery Mildew in Cucumis sativus

FENG XiangJun, WANG HongYu, YU Jing, CHI ChunYu, DING GuoHua

Scientia Agricultura Sinica. 2023, 56(14):  2701-2712.  doi:10.3864/j.issn.0578-1752.2023.14.006

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【Background】Cucumber (Cucumis sativus) is susceptible to many diseases, especially Fusarium wilt and powdery mildew. Although chemical control is effective, it is limited due to high residue and difficult degradation. Breeding cucumber varieties with broad-spectrum and long-lasting disease resistance is the fundamental strategy to solve this problem. Nonexpressor of pathogenesis-related genes 1 (NPR1) is a key regulator in systemic acquired resistance (SAR), which is involved in regulating the expression of a variety of defense-related genes and affecting plant disease resistance.【Objective】Overexpression of AtNPR1 in cucumber was used to explore the resistance of transgenic cucumber to Fusarium wilt and powdery mildew, and to provide experimental basis for breeding cucumber varieties with stronger and more lasting disease resistance.【Method】The AtNPR1 of Arabidopsis thaliana was cloned, the AtNPR1 overexpression vector was constructed, and the cucumber was transformed by Agrobacterium-mediated method to obtain transgenic cucumber plants with overexpression of AtNPR1. The expression levels of related defense genes in transgenic plants were determined by real-time fluorescence quantitative PCR (qRT-PCR) method. Transgenic plants of T₀ generation were selected for resistance identification of Fusarium wilt, and transgenic plants of T₁ generation were selected for resistance identification of powdery mildew. After inoculation transgenic plants with Fusarium oxysporum f. sp. cucumerinum and Golovinomyces cichoracearum, the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were quantified.【Result】Eight transgenic plants of the T₀ generation were successfully obtained, among which OE#4 and OE#5 exhibited high expression levels of AtNPR1, while OE#3 showed low expression levels. Analysis of the expression level of related defense genes in transgenic plants revealed that those plants overexpressing AtNPR1 exhibited stronger and faster expression of several defense genes. Furthermore, there was a positive correlation between the expression levels of defense genes and AtNPR1 in transgenic plants. Among them, the expression levels of PR1, PR4 and WRKY70 were significantly up-regulated. The results of disease resistance identification of transgenic plants showed that the transgenic plants exhibited more significant resistance, slower onset, mild symptoms, and significantly lower lesion area than wild type (WT) plants when subjected to the stress of Fusarium wilt and powdery mildew. The transgenic T₀ plants OE#4 and OE#5 exhibited no discernible lesions 3 days post-inoculation with F. oxysporum f. sp. cucumerinum, but displayed gray-brown lesions after 7 days without any signs of wilting. Conversely, the WT plants showed gray-brown lesions and slight wilting at 3 d post-inoculation, followed by severe leaf wilting at 7 d. After 7 days of inoculation with G. cichoracearum, both T₁ generation plants OE#2 and OE#7 as well as wild type (WT) plants exhibited lesions. However, the lesion area in OE#2 and OE#7 was significantly smaller than that in WT. After 15 days of inoculation, chlorosis appeared on the leaves of WT plants while the transgenic plants remained mildly affected. Compared with WT plants, transgenic plants exhibited lower MDA content and maintained higher levels of SOD, POD and CAT activities after inoculation. Additionally, the accumulation of ROS was less.【Conclusion】Overexpression of AtNPR1 in cucumber enhances its resistance against Fusarium wilt and powdery mildew.

Resistance Mechanism of Barnyard Grass (Echinochloa crus-galli) to Penoxsulam and Screening Herbicides for Its Control in Rice Fields

ZHANG LiYa, LI Qi, SHI ShanShan, MA YuMeng, LIU YaQi, ZHAO ChaoWei, WANG HeRu, CAO HaiQun, LIAO Min, ZHAO Ning

Scientia Agricultura Sinica. 2023, 56(14):  2713-2723.  doi:10.3864/j.issn.0578-1752.2023.14.007

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【Objective】Barnyard grass (Echinochloa crus-galli) is one of the main malignant weeds in rice fields in China. Acetolactate synthase (ALS) inhibitors, such as penoxsulam, are the main herbicides for controlling E. crus-galli in rice fields. Previously, our research team identified a suspected penoxsulam-resistant E. crus-galli population, AHTC-01, in the main rice production area of Tianchang City, Anhui Province, China. The objective of this study is to clarify its resistance levels to major herbicides, investigate the possible resistance molecular mechanisms, and to provide a theoretical basis for the effective control of resistant E. crus-galli and the delay of further development of herbicide resistance.【Method】Using the greenhouse potting method, the resistance levels of AHTC-01 to penoxsulam and its resistance patterns to different herbicides were determined at the whole-plant level. The target resistance molecular mechanism was explored through target gene sequencing and real-time quantitative PCR (RT-qPCR) analysis.【Result】Compared with the susceptible E. crus-galli population AHFY-01, the suspected resistant population AHTC-01 had developed high-level resistance to penoxsulam, with a resistance index (RI) of 620. Analysis of the target-site based resistance mechanisms showed that the ALS2 gene copy in the AHTC-01 population had a mutation from tryptophan (Trp) to leucine (Leu) at codon position 574, with a population mutation frequency of 100%. At 12 h after penoxsulam treatment, the relative expression level of ALS in the resistant E. crus-galli population AHTC-01 was 2.26 times of that in the susceptible E. crus-galli population AHFY-01. AHTC-01 also exhibited varying levels of cross-resistance to three other ALS inhibitors, bispyribac sodium, pyribenzoxim, and imazamox, with RIs of 8.24, 13.36, and 20.36, respectively. However, it remained susceptible to other herbicides with different modes of action (MOAs), including the acetyl-CoA carboxylase (ACCase) inhibitors cyhalofop-butyl, fenoxaprop-P-ethyl, and clethodim, the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor tripyrasulfone, and the synthetic auxin mimic florpyrauxifen-benzyl.【Conclusion】Mutation of the ALS2 at amino acid position 574 and ALS overexpression are one of the main reasons for the resistance of E. crus-galli population AHTC-01 to penoxsulam, which also confers cross-resistance to different ALS inhibitors. In actual agricultural production, effective control of this type of resistant E. crus-galli can still be achieved by rotating the use of other herbicides with different MOAs.

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

Spatial-Temporal Variation of Relative Yield Gap of Wheat and Maize and Its Response to Nitrogen Fertilizer in China

SHEN Zhe, HAN TianFu, QU XiaoLin, MA ChangBao, WANG HuiYing, LIU KaiLou, HUANG Jing, DU JiangXue, ZHANG Lu, LIU LiSheng, LI JiWen, ZHANG HuiMin

Scientia Agricultura Sinica. 2023, 56(14):  2724-2737.  doi:10.3864/j.issn.0578-1752.2023.14.008

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【Objective】This study aimed to explore the spatial-temporal variation characteristics and influencing factors of relative yield gap of wheat and maize in China during the past 15-20 years and the response of relative yield gap to nitrogen fertilizer under different soil productivity levels, so as to provide a theoretical basis for rational application of nitrogen fertilizer and the realization of high and stable yield of wheat and maize.【Method】Based on the long-term monitoring database, the difference of wheat and maize yield between fertilized area and non-fertilized area was used to represent the relative yield (RY). The highest relative yield (HRY), the average relative yield (ARY) and the relative yield gap (G_RY) were obtained by using the statistical of high-yielding households, the effects of fertilization and soil factors on the relative yield gap were determined used the random forest model, and soil productivity level was divided according to the yield of non-fertilized area. The relationship between the relative yield gap of wheat and maize and nitrogen application rate under different soil productivity levels was quantified.【Result】 HRY of wheat in China was 3.83-6.75 t·hm^-2, ARY was 2.10-3.42 t·hm^-2, and G_RY was 1.73-3.33 t·hm^-2, G_RY accounting for 44.64%-49.06% of HRY. HRY, ARY and G_RY of wheat were north China>middle-lower Yangtze Plain>northwest China>southwest China. HRY of maize in China was 6.53-8.20 t·hm^-2, ARY was 3.37-4.12 t·hm^-2, and G_RY was 3.16-4.08 t·hm^-2, G_RY accounting for 44.78%-50.52% of HRY. HRY, ARY and G_RY of maize were northeast China>north China>southwest China>northwest China. Except for north China, HRY and G_RY of wheat and maize increased with time. Except in northwest China, the G_RY decreased with the increase of nitrogen application rate in low and medium soil productivity, and the decrease amplitude was more significant in low soil productivity level, while the decrease of G_RY with nitrogen application rate in high soil productivity was not significant. Regionally, the balance points of nitrogen fertilizer application were found in wheat and maize in North China, wheat in middle-lower Yangtze Plain, and maize in northeast China at low and medium soil productivity. Overall, the nitrogen application rate and soil organic matter were relatively important influencing factors of G_RY for wheat and maize at low and medium soil productivity. Potassium application had a significant impact on the G_RY in middle-lower Yangtze Plain and north China, while organic matter had a significant impact on the G_RY in the northwest and southwest China under high soil productivity. 【Conclusion】N application and soil organic matter were important factors affecting the relative yield gap. The higher soil productivity level, the lower the effect of nitrogen fertilizer on reducing the relative yield gap. N fertilizer should be reduced appropriately in high productivity soil. In order to increase yield and avoid the waste of resource and environmental risks, it was suggested that the application rate of nitrogen fertilizer should not exceed its balance point. The recommended application rates of nitrogen fertilizer were 260.6 and 159.2 kg·hm^-2 for wheat and 262.5 and 246.0 kg·hm^-2 for maize at low and medium productivity levels in north China, respectively. In the middle-lower Yangtze Plain, 199.5 and 187.5 kg·hm^-2 were recommended for nitrogen application at low and medium productivity levels, respectively. In northeast China, the recommended amount of N fertilizer application was 259.5 and 228.0 kg·hm^-2, respectively. Under low and medium productivity levels in southwest and northwest China, N fertilizer should be appropriately increased. The potassium fertilizer reasonable application should be paid more attention at high soil productivity in north China and middle-lower Yangtze Plain. The improvement of soil organic matter should be as the main measures to achieve high and stable yields in southeast and southwest China.

Effects of Different Straw Returning Patterns on Soil CO₂ Emission and Carbon Balance in Maize Field

LI Jin, REN LiJun, LI XiaoYu, BI RunXue, JIN XinXin, YU Na, ZHANG YuLing, ZOU HongTao, ZHANG YuLong

Scientia Agricultura Sinica. 2023, 56(14):  2738-2750.  doi:10.3864/j.issn.0578-1752.2023.14.009

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【Objective】 The effects of different straw returning patterns on soil carbon dioxide (CO₂) emission characteristics and carbon balance were discussed, which provided a scientific basis for carbon (C) sequestration and emission reduction as well as the selection of straw returning patterns in Northeast China. 【Method】 A field micro-plot experiment were conducted with maize as the experimental crop, and three straw returning patterns were set up, including straw shallow returning (QH), straw deep returning (SH), and straw mulching (FG). No straw returning (CK) was used as the control treatment. The LI-8100A automatic soil C flux tester was used to monitor soil CO₂ emission characteristics under different straw returning patterns during the maize growth period. Effects of soil temperature, soil moisture content, pH, MBC, available nutrients and total nutrients of nitrogen, phosphorus and potassium on soil CO₂ emissions were analyzed, and soil carbon balance was investigated too. 【Result】 During the maize season, soil CO₂ emission rates showed a trend of first increasing and then decreasing under different straw returning patterns. The cumulative soil CO₂ emissions were as follows: FG>QH>SH>CK treatment. Compared with SH treatment, the cumulative soil CO₂ emissions under FG and QH treatments increased by 14.0% and 6.4%, respectively. There was a significant difference between the treatments (P<0.05). The single factor model fitting of soil CO₂ emission rates, soil temperature and soil moisture content under different straw returning patterns showed a quadratic function correlation, and reached a significant level (P<0.05), soil temperature could explain the variation of soil CO₂ emission rate of 68.2%-73.7%, and soil moisture content could explain 21.3%-37.5%. However, the two-factor composite model of soil temperature and soil moisture content could better explain the variation of soil CO₂ emission rate, with an explanation of 78.5%-82.8%. Correlation analysis showed that cumulative CO₂ emissions were significantly correlated with available potassium and MBC (P<0.01), and significantly correlated with soil organic matter, available nitrogen, total nitrogen, and pH (P<0.05). The soil carbon balance was positive under different straw returning patterns, which were the "sink" of atmospheric carbon dioxide. The soil carbon balance and carbon sequestration potential under the SH treatment were significantly higher than the QH and FG treatments by increased of 23.4%, 475.7% and 7.1%, 30.7% (P<0.05), respectively. Compared with other treatments, the SH treatment showed a strong carbon "sink" function. In the two-year harvest period, straw returning treatments significantly increased maize yield, SH treatment had the highest maize yield, but there was no significant difference with QH and FG treatments. 【Conclusion】 Therefore, under the conditions of this experiment, taking into account the carbon sequestration and emission reduction effect and yield, SH was a better straw returning pattern compared with the three patterns.

Rapid Identification for Bradyrhizobium japonicum 5873 by PCR and Its Evaluation in Application

MA MingChao, JIANG Xin, WANG PengHui, GUAN DaWei, LI Jun

Scientia Agricultura Sinica. 2023, 56(14):  2751-2760.  doi:10.3864/j.issn.0578-1752.2023.14.010

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【Objective】Bradyrhizobium japonicum is one of the important functional bacteria in microbial fertilizer products. As a commercialized strain, B. japonicum 5873 was wildly used in agricultural production and played important roles in symbiotic nitrogen fixation. Therefore, it is necessary to establish a rapid identification method at the strain level by screening and identifying specific primers for rhizobia, and is of great importance in the aspects of microbial fertilizer product quality inspection, strain identification and function evaluation.【Method】According to the whole genome sequencing of B. japonicum 5873 and other strain related sequences from NCBI, as well as the differential fragments of B. japonicum USDA 6^T which is highly homologous to B. japonicum 5873 (the ANI value of the genome is greater than 99.95%), specific PCR primers were designed and obtained. After optimization of PCR reaction conditions and the detection of sensitivity and specificity, a rapid detection method for B. japonicum 5873 was established. Then, using pot experiment, B. japonicum 5873 was mixed with other rhizobium strains and inoculated in the soybean rhizosphere, and the above method was used to evaluate the competitive nodulation ability of B. japonicum 5873.【Result】A set of species-specific primers 4-4 and Q1 (4-4-F 5′-GATAAGGCCACGGGTGAACA-3′/4-4-R 5′-CACTCGATAAGCTCCGCTGT-3′ and Q1-F 5′-CCGGTCGTGACTGGAATGAT-3′/Q1-R 5′-TCGAGGCCTACAAGAACGTC -3′), were successfully designed to selectively amplify 355 and 218 bp amplicon from B. japonicum 5873. Well specificity was demonstrated by reference strains, from which only the targeted bands of B. japonicum 5873 were observed. Amplifications were performed in a 25 μL reaction mixture, which contained Premix Taq^TM 12.5 μL, template DNA (15 ng of genomic DNA), primers 4-4 and Q1 (1.0 μL, respectively). The PCR cycling consisted of one cycle of 5 min at 95 ℃ and 30 cycles of 45 s at 94 ℃, 45 s at 61 ℃, and 60 s at 72 ℃. A final extension step was run for 10 min at 72 ℃. The sensitivity was 1 850 CFU/µL. In addition, the method mentioned above was successfully used to evaluate the competitive nodulation ability of B. japonicum 5873, which was consistent with the results of traditional BOX-PCR identification.【Conclusion】The established rapid detection method can directly use the bacterial solution or squeezed nodules as templates, which will eliminate laborious tasks, such as processes of nodule isolation, purification, culture, DNA extraction, sequencing and sequence comparison. It only takes a few hours to accurately detect B. japonicum 5873, which provides a reference for product quality detection and competitive nodulation ability evaluation of microbial fertilizers.

HORTICULTURE

Screening and Comprehensive Evaluation of Tomato Rootstocks with High Efficiency of Phosphorus Utilization

GAO ZiYuan, HU JingAng, ZHANG BeiBei, GONG Biao

Scientia Agricultura Sinica. 2023, 56(14):  2761-2775.  doi:10.3864/j.issn.0578-1752.2023.14.011

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【Objective】Phosphorus is a non-renewable resource, and the uptake and utilization efficiency of soil phosphorus by plants is low. It is of great economic and ecological value to improve the efficiency of phosphorus absorption and utilization by grafting. The aim of this study was to screen the varieties of grafting tomato rootstocks with efficient use of phosphorus and to establish a light, simple and efficient evaluation technology system, which had theoretical and practical guiding effects on the breeding, demonstration and promotion of new rootstocks with efficient use of phosphorus.【Method】The scion tomato varieties of Qinglove No.1 were grafted on 25 rootstocks (G1-G25) or self-grafted (G0). The experiment was conducted in tomato seedling stage and whole growth stage. In the seedling stage test, the grafted seedlings were treated with normal phosphorus (Hoagland nutrient solution, NP) and low phosphorus (Hoagland nutrient solution with 10% phosphorus content, LP) by hydroponics. 15 days later, 16 indexes were measured, including growth and development, phosphorus absorption and utilization efficiency of grafted seedlings. In the whole growth period experiment, the grafted seedlings were still used as the test material. The phosphorus application level of the control group was set at 1 272 kg∙hm^-2, and that of the LP treatment group was 50% of that of the control group. Nine indexes such as fresh weight of stem and leaf, yield and fruit quality were determined. Then, the correlation analysis, principal component analysis, membership function value analysis, cluster analysis, multiple regression analysis and other mathematical analysis models were used to screen the test materials with phosphorus efficient utilization type grafting tomato rootstock and to evaluate them comprehensively.【Result】The average coefficient of variation of indexes at seedling stage and whole growth stage under NP treatment was 9.74% and 2.85%, respectively, and the average coefficient of variation of indexes at seedling stage and whole growth stage under LP treatment was 16.10% and 5.84%, respectively. The variation coefficient of each index under LP treatment was generally higher than that under NP treatment, indicating that the difference of influence of rootstock genotype on grafted tomato was amplified under LP condition. Correlation analysis showed that yield (I-17) under NP condition was positively correlated with stem and leaf dry weight (I-1), stem diameter (I-5), stem and leaf P mass fraction (I-7), stem and leaf P absorption efficiency (I-9), whole plant P absorption efficiency (I-11), and stem and leaf P transport efficiency (I-12); under the LP condition, the tomato yield (I-17) was positively correlated with stem and leaf dry weight (I-1), root dry weight (I-2), stem diameter (I-5), strong seedling index (I-6), stem and leaf P mass fraction (I-7), stem and leaf P absorption efficiency (I-9), root and leaf P absorption efficiency (I-10), whole plant P absorption efficiency (I-11), and stem and leaf P transport efficiency (I-12). The ranking of principal component and membership function values showed that the ranking rules of the two analysis methods were basically consistent, and both of them were consistent with the performance pattern of cluster analysis. However, there were slight differences in the ranking of individual rootstock varieties, so this paper used the comprehensive average performance of the two rankings as the final ranking, and calculated the top five grafting combinations (G24, G1, G8, G3, and G25). Through multiple regression analysis, the seedling stage evaluation indexes suitable for low phosphorus tolerance of tomato rootstock were obtained, and the regression equations of key seedling stage indexes affecting yield and quality were established: Y_I-17=1354.630-5.552X_I-4, Y_I-20=2.956X_I-5-7.949X_I-14+2.927, and Y_I-23=48.807+0.005X_I-11.【Conclusion】In this study, a set of simple, objective and relatively objective technology system for screening and comprehensive evaluation of tomato rootstock with efficient utilization of phosphorus was established. Three rootstocks, including Korean rootstock No. 1, Jinpeng Rootstock No. 1 and Western Tomato rootstock, were identified to have comprehensive advantages of efficient utilization of phosphorus fertilizer.

Mechanism Underlying the Improved Quality of Bagged Fruits in Ponkan

SHI Ying, CHEN SiYi, ZENG YiKe, TANG Jun, LI DiPing, LI GuoJing, HUANG XianBiao, LI ChunLong, XIE ZongZhou, LIU JiHong

Scientia Agricultura Sinica. 2023, 56(14):  2776-2786.  doi:10.3864/j.issn.0578-1752.2023.14.012

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【Background】 Ponkan (Citrus reticulata) is an important loose-skin tangerine in China. Egan No. 1 Ponkan is widely grown in Dangyang City, Hubei Province. When this cultivar is used for producing fruit, the major problem is that advanced harvesting leads to undesirable fruit quality, and thus, to a negative impact on consumer appeal. Therefore, the industry has a critical need for the technologies that allow growers to delay harvest until Egan No. 1 Ponkan fruit is fully mature. 【Objective】 In this study, whether bagging improves the quality of fruit produced by Egan No. 1 Ponkan was tested, and the underlying mechanism by examining the expression levels of genes involved in sucrose and citric acid metabolism was analyzed. 【Method】The experiment was conducted in orchards that produce Egan No. 1 located in Banyue Town, Dangyang City, Hubei Province, to compare the qualities of fruit that were produced after bagging to control fruit that was not bagged. The indexes were compared, including peel brightness (L*), color parameters a*, b*, citrus color index (CCI), total soluble solids, titratable acids, soluble sugars and organic acids. In addition, qRT-PCR was used to analyze the expression levels of the genes associated with sucrose and citric acid metabolism. 【Result】The bagging induced changes in peel color parameters, including changes in a*, b*, CCI, increased L*, and significantly reduced peel firmness. The bagging significantly increased the total soluble solid content when the fruit was harvested, and the sucrose, fructose and glucose levels were significantly elevated. A gas chromatography analysis showed that the levels of citric acid, the major organic acid in citrus fruits, was slightly higher in the bagged fruits relative to the control fruit. The qRT-PCR analysis showed that the expression levels of three genes associated with sucrose biosynthesis (CsSPS3, CsSPS4 and CsSS2) were significantly higher in the bagged fruit relative to the control fruit. In contrast, the expression levels of genes implicated in citric acid catabolism, such as CsACO1, were remarkably downregulated in the bagged fruit. 【Conclusion】Taken together, these data indicated that the bagging improved the quality of Ponkan fruit possibly by changing the ambient temperature and humidity in the micro-milieu of the bagged fruits, which might have elevated the expression levels of genes that influence soluble sugar content and led to better sugar accumulation and improved fruit quality of Ponkan.

FOOD SCIENCE AND ENGINEERING

Influencing Factors of Gluten Network Structure Analysis Using Confocal Laser Scanning Microscopy

JIANG JiKai, ZHANG YingQuan, GUO BoLi, YANG JingJie, HUANG LuYao, LI Ming, ZHONG Geng

Scientia Agricultura Sinica. 2023, 56(14):  2787-2797.  doi:10.3864/j.issn.0578-1752.2023.14.013

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【Objective】The thickness of sample slices, the concentration of staining solution and the staining time are the key factors influencing the results of gluten network structure analysis using confocal laser scanning microscopy (CLSM). The aim of this study was to investigate the effects of the slice thickness, the staining solution concentration and the staining time on the CLSM results of gluten network structure, and the optimal slice thickness and staining conditions for the dough structure analysis were selected, so as to provide a technical support for the studies on dough structure and/or its final products. 【Method】Two wheat varieties, i.e., Zhengmai 366 (Z366) and Xiaoyan 22 (X22) with different wet gluten indexes, were selected as raw materials, which were milled and made into dough, respectively. Then, the dough samples were frozen for 0 and 1 day. The dough slice thickness was set as 10, 14 and 20 μm, respectively. Meanwhile, two concentrations of staining solution (0.001%+0.01%, 0.025%+0.25%) using Rhodamine B (RDB) and fluorescein isothiocyanate (FITC) were prepared. The staining time was set for 1, 10 and 20 min, respectively. 【Result】The sample slice with 10 μm thickness showed the best and clearest image for both doughs of Z366 and X22. Besides, under this slice thickness, the gluten network of the dough frozen stored for 0 day showed the most obvious difference compared with that for 1 day (P<0.01). Additionally, under low-concentration staining agent (0.001%+0.01%), the clearer the results of gluten network structure were obtained with longer staining time. Oppositely, under the high-concentration staining agent (0.025%+0.25%), the slice with a staining time of 1 min showed the most rational results on the gluten network structure, whereas the result on the slice with a staining time of 10 min was the worst. 【Conclusion】The gluten network structure of dough with different protein content and freezing treatments demonstrated more differences when the thickness of dough slices was 10 μm, indicating the gluten network structure of different samples could be distinguished obviously. Under the selected thickness, the gluten network structure of 20 min stained with low-concentration mixed staining agent showed the best effect if time-consuming was not considered. Moreover, the high-concentration staining for 1 min was time-saving and better for observing the structure of starch granules.

ANIMAL SCIENCE·VETERINARY SCIENCE

Population Structure and Genetic Diversity of Lufeng Cattle and Leiqiong Cattle Based on Genome-Wide SNPs

TONG Xiong, LUO Wei, MIN Li, ZHANG ZhiFei, MA XinYan, LUO ChengLong, CHEN WeiDong, XU Bin, LI DaGang

Scientia Agricultura Sinica. 2023, 56(14):  2798-2811.  doi:10.3864/j.issn.0578-1752.2023.14.014

Abstract ( 178 )   HTML ( 12 )   PDF (3864KB) ( 568 )   Save

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【Objective】Phylogenetic relationship among Lufeng cattle, Leiqiong cattle and domestic cattle in different regions worldwide, and genetic diversity of different domestic cattle populations were studied, so as to lay a theoretical foundation for the identification and protection of domestic cattle resources. 【Method】Tissue samples of 12 individuals of Lufeng cattle and 17 ones of Leiqiong cattle were collected for whole genome resequencing. Combined with another 92 cattle genomes from 24 breeds worldwide available in the NCBI database, a panel of cattle genomes comprising 121 individuals were generated from 25 breeds to carry out population genetics study. Bos taurus ARS-UCD1.2 assembly was selected as the reference genome. High-quality reads were obtained by genome alignment and quality control. Genomic SNPs were detected by GATK software. Population structure was analyzed by phylogenetic tree construction, PCA clustering, and Admixture evaluation. Genetic diversity of the populations was studied by estimating nucleotide diversity (Pi), heterozygosity (Hp), and linkage disequilibrium (LD). 【Result】A total of 6 905 944 306 clean reads were obtained by genome sequencing from 29 individuals of the two cattle breeds in Guangdong. Average genome coverage and average sequencing depth of each sample were 97.99% and 12.78×, respectively. After integrating the NCBI cattle genome data, 14 664 391 population SNPs were identified. The results of phylogenetic tree, PCA and Admixture showed that a primary division was found between cattle from taurine and indicine. Moreover, indicine cattle split on Chinese and Indian cattle, and Northeast Asian cattle (Hanwoo and Yanbian) and Tibetan cattle separated from European taurine group, while Wenling cattle and Zhoushan cattle differentiated from Chinese indicine group. Lufeng cattle and Leiqiong cattle belonged to pure Chinese indicine cattle. Lufeng cattle and Wannan cattle, Leiqiong cattle and Ji'an cattle showed the closest relationship, respectively. The relationships indicated that Lufeng cattle and Leiqiong cattle in the adjacent areas belonged to two independent breeds. Some Lufeng and Leiqiong individuals were genetically admixed with European taurine and Northeast Asian taurine cattle, and the admixed proportion was high, indicating that these two breeds needed to strengthen the purification and rejuvenation within the population. Compared with European taurine cattle and Korean cattle, for Chinese domestic cattle, LD decay rate was faster, and nucleotide diversity (Pi) and heterozygosity (Hp) were higher, indicating that genetic diversity of Chinese domestic cattle was richer. Compared with other Chinese domestic cattle, LD levels of Lufeng cattle and Leiqiong cattle were lower, heterozygosity (Hp) was higher, and the density distribution of nucleotide diversity (Pi) and heterozygosity (Hp) was more concentrated, indicating that the two cattle breeds were less subject to artificial selection and maintain higher genetic diversity. 【Conclusion】Population structure and genetic diversity of Lufeng cattle and Leiqiong cattle were analyzed by genome-wide SNPs, which provided data support for independent classification and conservation of these two cattle breeds.

Effects of Caragana korshinskii Kom. on Serum Indexes, Rumen and Colon Microbiota of Tan Sheep

HAN Jing, WANG XiaoQi, DUAN ZiYuan

Scientia Agricultura Sinica. 2023, 56(14):  2812-2827.  doi:10.3864/j.issn.0578-1752.2023.14.015

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【Objective】 This study aimed to examine the effects of Caragana korshinskii Kom. (CK) at the rate of 10% of the diet on blood lipids, immune indicators, and rumen and colon microbiota of Tan sheep, so as to provide a theoretical basis for CK being partial Tan sheep forage. 【Method】 Twelve Tan sheep (4-month-old) with an initial average weight of 27.5 ± 3.32 kg were randomly divided into 2 groups (6 in each group): NC group was fed with 30% concentrate and 70% alfalfa; CK group, with 30% concentrate, 60% alfalfa and 10% CK, and drinking water ad libitum. The nutrient composition of the two groups were similar, but the fiber content of CK was slightly higher than that of alfalfa. The preparatory experiment was in 10 days and the formal experiment was lasted for 60 days. At the end of the 60 days, jugular vein blood was collected to measure blood biochemical indexes (blood lipids and immune factors) and rumen fluid, and colon content were collected to detect the changes of flora in rumen and colon, respectively, by 16S rRNA sequencing method. Through construction of the recombinant plasmid, such as Selenomona, Prevotella, Blautia, Desulfovibrio, Ruminococcus and Bacteroides, the absolute quantitative of important genera were detected by real-time quantitative PCR (qPCR) comparing to its standard curve, respectively. 【Result】 At the end of the trial period, the end weight and daily average feed intake of Tan sheep were similar between two groups. Compared with NC group, the indexes related to the lipid absorption and digestion in CK group decreased, such as the content of serum triglyceride (TRIG, P<0.05) and very low density lipoprotein (VLDL, P<0.01) decreased markedly, while the concentration of low density lipoprotein (LDL) tended to decrease significantly (0.05<P<0.1). The inflammatory and lipolytic factor, such as interleukin-6 (IL-6), increased significantly (P<0.05) whereas the inflammatory factor, γ-Interferon (IFN-γ) marked decreased tendentiously (P=0.058). By analyzing microflora of rumen fluid and colon contents, adding CK to diet increased community Shannon diversity index of colonic bacteria communities significantly (P<0.01), while tended to decrease the Chao1 index in rumen (0.05<P<0.1). Compared with NC group, the relative abundance of Oscillospira under CK decreased (P=0.07), while that of Akkermansia notable under CK increased (P<0.05) in rumen. The relative abundance of harmful bacteria, like Treponema, and carbohydrate degradation related Bacteroides significantly decreased (P=0.059, P=0.061), but that of Oscillospira, which a butyric acid producing bacteria related to obesity, constipation and immunity, gained markedly (P<0.05) in colon under CK. It was evidently deduced that the bacterial communities in rumen and colon had significant difference on the capacities for lipid metabolism and biosynthesis of other secondary metabolite between two groups (P<0.05). Based on Pearson correlation analysis, it was showed that the level of serum TRIG was positively correlated with rumen and colonic Pseudobutyrivibrio (P<0.05), which was both fiber degrading bacteria and butyric acid producing bacteria. 【Conclusion】 There was no significant effect of CK partial substitution for alfalfa on the growth performance of Tan sheep, but could reduce the concentrations of serum TRIG, LDL and VLDL through affecting the gastrointestinal tract microbiota, and then participate in the lipid metabolism of the host.

Identification of Key Amino Acids in the Antigenic Variation of Eurasian Avian-Like H1N1 Swine Influenza Viruses

ZHANG NaiXin, XU ChengZhi, YANG YuYing, ZHANG YaPing, WAN YunFei, QIAO ChuanLing, CHEN HuaLan

Scientia Agricultura Sinica. 2023, 56(14):  2828-2836.  doi:10.3864/j.issn.0578-1752.2023.14.016

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【Background】 The antigenicity of influenza virus is mainly determined by the hemagglutinin (HA), a surface glycoprotein of the virus. Our previous study indicated that amino acid changes at positions 190, 230 and 269 (H3 numbering) of HA protein resulted in antigenic escape by using the monoclonal antibody (mAb) against the HA protein of Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV). These three amino acid substitutions widely existed in the HA protein of the recently isolated EA H1N1 SIVs. 【Objective】This study aimed to explore which amino acids played a key role in the antigenicity of the virus, and further provide scientific basis for the control of influenza. 【Method】 In this study, A/swine /Liaoning /SY72/2018 (H1N1) (SY72) was selected as the model virus, and its reverse genetic system (RGS) was established. Then, using SY72 virus as backbone, three reassortant viruses were rescued by introducing the respective single amino acid mutation at position 190, 230, and 269 into HA protein. Antisera were raised by inoculating the rSY72-inactivated vaccine into specific-pathogen-free (SPF) chickens and non-immunized pigs. The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the rSY72 virus. Then their effects on viral replication capacities and receptor binding properties were further evaluated. 【Result】 Sequence analysis showed that the HA of SY72 virus carried 190D, 230M, and 269R, respectively. The viruses, including rSY72, rSY72HA/D190N, rSY72HA/M230I, and rSY72HA/R269M, were rescued by RGS. The results of neutralization test showed that all the three mutant viruses could react with two mAbs, to some extent, compared with the rSY72 virus. The HI results indicated that the HI antibody titers of the rSY72HA/D190N reacted with the rSY72-immunized chicken and pig sera were 4- and 8-fold lower than those of the rSY72 virus with the respective sera. However, the rSY72HA/M230I and rSY72HA/R269M virus reacted well with these two sera. The results indicated that residue 190 in the HA had the important effects on the viral antigenicity. Results of the viral plaque assay and growth curve experiments demonstrated that plaque sizes generated by the rSY72HA/D190N virus were smaller than those by the rSY72 virus in MDCK cells. The replication ability of the rSY72HA/R269M virus was significantly decreased in MDCK cells, compared with that of the rSY72 virus. Furthermore, the three amino acid mutations had no impact on the viral receptor-binding preference. 【Conclusion】 The amino acid at position 190 of HA protein played an important role in determining the antigenicity of EA H1N1 SIV. The mutation of amino acid at position 269 reduced the viral replication ability in MDCK cells. These results suggested that more attentions should be paid for monitoring these residue changes in the influenza surveillance, so as to improve early warning of influenza in animals.