Scientia Agricultura Sinica

Screening of Core Markers and Construction of DNA Fingerprints of Semi-Waxy Japonica Rice Varieties

ZHAO ChunFang,ZHAO QingYong,LÜ YuanDa,CHEN Tao,YAO Shu,ZHAO Ling,ZHOU LiHui,LIANG WenHua,ZHU Zhen,WANG CaiLin,ZHANG YaDong

Scientia Agricultura Sinica. 2022, 55(23): 4567-4582. doi:10.3864/j.issn.0578-1752.2022.23.001

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【Objective】 A set of variety DNA fingerprint identification system based on the core markers of genes regulating rice important traits was constructed, which will establish a foundation for strengthening the germplasm management and protection of the mainly promoted semi-waxy japonica rice varieties with high eating quality.【Method】 34 semi-waxy japonica rice varieties mainly cultivated in Jiangsu, Zhejiang and Shanghai were used as the test materials. The key differential sequence sites in genes regulating rice important traits were screened and core SNP or InDel markers were developed through multiple methods such as polymorphism testing of existing markers, gene sequence alignment from public databases and genome resequencing. SNP markers were developed into simple PCR markers based on electrophoretic bands by As-PCR technology. Genotype information was obtained by electrophoretic band characterization and type analysis, and the DNA fingerprint database of the semi-waxy japonica rice varieties was constructed..【Result】 54 core markers derived from 40 key genes regulating rice important traits were obtained, including 18 SNP and 36 InDel markers; 155 characteristic and effective bands were identified by 54 markers in the tested rice varieties, which were transformed into 155 0/1 data sites. The DNA fingerprint database of each variety was established and could distinguish it from all tested varieties. Genetic diversity analysis showed that the variation range of genetic similarity among varieties was 0.47-0.90, among which the lowest similarity coefficient was detected between Nanjing 7718 and Suxiangjing 100, while the highest similarity coefficient was detected between Nanjing 9308 and Nanjing 9036, among which there were 8 differential data sites. Genetic relationship analysis indicated that 34 varieties were divided into 6 branches, of which Nanjing 7718 was an independent branch, suggesting it has a distant relationship from other varieties. Further verification of the identification effect of core markers showed that the set of markers could effectively distinguish 14 new semi-waxy japonica rice varieties. The cluster diagram showed that they were distributed in three groups of Ⅱ, Ⅲ and Ⅳ, confirming the differences of genotype information among varieties; using this set of markers, the authenticity of an unknown semi-waxy rice variety was also identified. According to genotype and cluster analysis, it was determined as Nanjing 9108.【Conclusion】 After optimization and screening, 54 core markers that could accurately distinguish all the tested semi-waxy japonica varieties were obtained, and developed into simple PCR markers detected by electrophoresis. Using this set of marker combinations, the DNA fingerprints of 34 semi-waxy japonica varieties in Jiangsu, Zhejiang and Shanghai were constructed.

Identification of Wheat Circular RNAs Responsive to Drought Stress

LI Ning,LIU Kun,LIU TongTong,SHI YuGang,WANG ShuGuang,YANG JinWen,SUN DaiZhen

Scientia Agricultura Sinica. 2022, 55(23): 4583-4599. doi:10.3864/j.issn.0578-1752.2022.23.002

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【Objective】 Drought is one of the foremost abiotic stress limiting global wheat production. Exploring the molecular mechanism of wheat response to drought stress have great significance in wheat molecular breeding. Circular RNAs (circRNAs) have been proved to play an important role in the process of plants tolerance to environmental stresses. Therefore, identifying circRNAs involved in drought stress response will help to construct a regulatory network of wheat drought tolerance, and lay a foundation for analyzing the drought resistance mechanism in wheat. 【Method】 In this study, two wheat varieties (Zhoumai13 and Jimai38) with significant differences in drought resistance were used and circRNA-seq was performed on their root samples under well-watered and drought conditions. Differentially expressed circRNAs related to drought stress response were screened based on the identified circRNAs and their microRNAs (miRNAs) targets were predicted. Further, potential circRNA-miRNA-mRNA regulatory modules related to wheat drought stress response were constructed according to the expression patterns of miRNAs and their target genes under drought stress..【Result】 A total of 1 409 wheat circRNAs were identified, most of which (68.91%) were exonic circRNAs. Only 133 circRNAs were simultaneously identified in both varieties. A total of 239 differentially expressed circRNAs were identified under drought stress, of which 138 circRNAs were specifically differentially expressed in the drought-resistant variety Zhoumai 13 (ZM13), and 19 circRNAs were differentially expressed simultaneously in both varieties. Besides, 34 targeted miRNAs and 1 408 miRNA target genes were predicted. Based on the expression patterns of these differentially expressed circRNAs, targeted miRNAs, and miRNA target genes, five potential circRNA-miRNA-mRNA regulatory modules centered on tae-miR9664-3p, tae-miR1122b-3p, tae-miR9662a-3p, tae-miR6197-5p and tae-miR1120c-5p in response to drought stress were screened..【Conclusion】 Wheat circRNAs have obvious specificity in different cultivars and different expression patterns among different drought-tolerant wheat cultivars. A total of 239 wheat circRNAs and 5 potential circRNA-miRNA-mRNA regulatory modules in response to drought stress were identified in the present study.

The Comparison of Ammonia Volatilization Loss in Winter Wheat- Summer Maize Rotation System with Long-Term Different Fertilization Measures

YI YingJie, HAN Kun, ZHAO Bin, LIU GuoLi, LIN DianXu, CHEN GuoQiang, REN Hao, ZHANG JiWang, REN BaiZhao, LIU Peng

Scientia Agricultura Sinica. 2022, 55(23): 4600-4613. doi:10.3864/j.issn.0578-1752.2022.23.003

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【Objective】 Based on a long-term positioning test platform, the differences of annual ammonia volatilization loss of soil nitrogen in wheat-maize rotation system under different fertilization treatments were compared, aimed to provide a theoretical basis for reducing nitrogen volatilization loss and improving fertilizer use efficiency.【Method】 From 2019 to 2021, the winter wheat variety Shimai 15 and summer maize variety Zhengdan 958 were used as experimental materials in this study. With no nitrogen fertilizer applied as the control (CK), the two kinds of nitrogen fertilizer types (organic manure: M; urea: U) and two N application rates (380 kg N·hm^-2: M1, U1, U2M2, and 190 kg N·hm^-2: U2, M2) were set, and the distribution of N fertilizer between two crops was 47.4% for wheat and 52.6% for maize. The venting method was used to compare the differences in annual ammonia volatilization rate, cumulative loss, grain yield, and nitrogen use efficiency in a wheat-maize rotation system under different fertilization treatments.【Result】 Different fertilization treatments significantly affected soil ammonia volatilization in the two growing seasons. The soil ammonia volatilization mainly occurred 0-7 days after fertilization during the annual year in the wheat and maize crops, and the difference between the treatments gradually became smaller. The annual ammonia volatilization loss ranged from 8.6 to 79.4 kg N·hm^-2. The highest ammonia volatilization loss was 79.4 kg N·hm^-2 under U1 treatment, which was 18.5%, 111.7%, 162.3%, 20.5%, and 825.7% higher than that under U2, U2M2, M1, M2, and CK, respectively. The high nitrogen application rate increased soil ammonia volatilization loss, and the loss of inorganic nitrogen fertilizers was greater than that of organic fertilizers. The annual nitrogen fertilizer ammonia volatilization loss rate under U2M2, M1, and M2 treatment was 80.9%, 40.8%, and 61.3% lower than that under U1, respectively. It was indicated that the organic and inorganic fertilizers combined application or single organic fertilizers application could significantly reduce ammonia volatilization losses. The annual grain yield under U2M2 treatment was the highest, which was 24 621.8 kg·hm^-2, and was 10.1%, 24.7%, 11.7%, and 32.7% higher than that under U1, U2, M1, and M2, respectively. The annual nitrogen utilization efficiency under U2M2 treatment was 52.6%, which was 11.3%, 4.1%, 13.4%, and 10.7% higher than that under U1, U2, M1, and M2, respectively. U2M2 treatment reduced the ammonia volatilization loss and simultaneously increased the grain yield and nitrogen use efficiency, which was an ideal fertilization strategy for the annual rotation of winter wheat and maize.【Conclusion】 The application of organic fertilizer could significantly reduce the annual ammonia volatilization loss of the wheat-maize rotation system, and increase the annual grain yield and nitrogen fertilizer use efficiency. Considering the source of organic fertilizer and the convenience of application, the organic and inorganic fertilizers combined application could be used as the main fertilization method to reduce the loss of ammonia volatilization and to improve the efficiency of nitrogen fertilizer use efficiency in wheat-maize rotation production system.

Cry1Ac Protein Content Responses to Alternating High Temperature Regime and Drought and Its Physiological Mechanism in Bt Cotton

YIN YanYu, XING YuTong, WU TianFan, WANG LiYan, ZHAO ZiXu, HU TianRan, CHEN Yuan, CHEN Yuan, CHEN DeHua, ZHANG Xiang

Scientia Agricultura Sinica. 2022, 55(23): 4614-4625. doi:10.3864/j.issn.0578-1752.2022.23.004

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【Objective】 This study was conducted to investigate the effects of alternating high temperature and drought on the content of Cry1Ac protein in Bt cotton and the underlying physiological mechanism, so as to provide a reference for the safe and stable utilization of insect resistance of Bt cotton in production.【Method】 The conventional cultivar Sikang 1 (SK-1) and hybrid cultivar Sikang 3 (SK-3) were used in 2019 and 2020 in experimentally controlled greenhouse, Yangzhou University. From 7:00 am to 7:00 pm, two high-temperature treatments [34℃ (A1) and 38℃ (A2)] were imposed on cotton plants, followed by an optimum temperature 28℃ during the remaining night hours. There were two treatments for soil moisture content, which were 50% (B1) and 60% (B2) field capacity. The treatment with 32℃/28℃ and 75% field capacity was set as the control (CK). The leaf Cry1Ac protein content and its physiological mechanism were detected on 4, 7, and 10 days after stress (DAS), respectively.【Result】 Compared with CK, the Cry1Ac protein content all decreased under the stresses of high temperature and drought, and with the extension of the stress time, greater decrease was observed. The extent of decline for A1B2 was the smallest, followed by A1B1, while A2B1 and A2B1 were the largest. The Cry1Ac protein content under A1B2 was significantly lower than CK after 7 DAS, while the significant differences between A1B1, A2B2, A2B2 and CK were detected after 4 DAS. The Bt gene expression level, soluble protein (SP), free amino acid (aa) contents, nitrate reductase (NR), the glutamic pyruvic transaminase (GPT), glutamic oxaloacetate transaminase (GOT), glutamine synthetase (GS) and glutamate synthase (GOGAT) activities showed a downward trend. While the tannin content, the activities of protease and peptidase showed an upward trend. The correlation analysis and path analysis showed SP, aa, NR, GPT, GOT, GS, and GOGAT were positively correlated with Cry1Ac protein content. The tannin content, activities of protease, and peptidase were negatively correlated with Cry1Ac protein content. NR, GPT and GS could be key indices for the Cry1Ac protein content.【Conclusion】 The interaction of high temperature and drought resulted in the decrease of Cry1Ac protein content in Bt cotton, with greater decrease observed as the stress extended. There was no significant difference between the treatment with 34℃/28℃ and 60% field capacity and CK in 7-10 DAS. The reduction extent of Cry1Ac for alternating high temperature regime and drought decreased and the period was delayed. NR, GPT and GS could be key indices for the Cry1Ac protein content.

Functional Analysis of the Interaction Between Transcription Factors VqWRKY6 and VqbZIP1 in Regulating the Resistance to Powdery Mildew in Chinese Wild Vitis quinquangularis

ZHANG Jie, JIANG ChangYue, WANG YueJin

Scientia Agricultura Sinica. 2022, 55(23): 4626-4639. doi:10.3864/j.issn.0578-1752.2022.23.005

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【Objective】 As the world’s main cultured varieties, Vitis vinifera has the advantages of high yield and good quality, however, the resistance of V. vinifera to disease is poor. Powdery mildew is a fungal disease that seriously endangers viticulture. The resources of Chinese wild grape are abundant, which can provide sufficient germplasm resources for disease-resistant breeding. This objective of this study is to screen grape transcription factor genes that regulate the resistance to powdery mildew, and to explore the mechanism of these genes regulating the resistance to powdery mildew, which can provide high-quality genetic resources for selection and breeding of grape disease-resistant varieties.【Method】 VqWRKY6 was cloned from Chinese wild Vitis quinquangularis ‘Shang-24’. The sequences were analyzed using DANMAN and MEGA-X software and subcellular localization analysis was adopted using Arabidopsis thaliana protoplasts by PEG-mediated transformation, which was performed for the location of transcriptional regulation. The yeast two-hybrid and bimolecular fluorescence complementation were adopted to prove that VqWRKY6 can interact with the transcription factor VqbZIP1 to form a transcriptional complex. Taking the leaves of the disease-susceptible grape ‘Cabernet Sauvignon’ as the material, the transient transformation mediated by Agrobacterium was performed in the leaves of ‘Cabernet Sauvignon’. After Uncinula necator inoculation on the leaves, the pathogenesis symptoms were observed, hyphal development progression was microscopically visualized with trypan blue staining, and reactive oxygen species (ROS) accumulation was visualized using DAB staining. The differences between grape leaves co-overexpressing of VqWRKY6 and VqbZIP1, overexpressing of VqWRKY6 alone, overexpressing of VqbZIP1 alone, and the control group were compared. The qRT-PCR was used to analyze the expression level of anti-disease genes under U. necator induction.【Result】 VqWRKY6 is located on grape chromosome 2, encodes 342 amino acids and belongs to the group Ⅲ subfamily of the WRKY family. VqWRKY6 exerts a transcriptional regulatory function in nucleus. The propagation rate of U. necator on the surface of ‘Cabernet Sauvignon’ leaves after co-overexpressing of VqWRKY6 and VqbZIP1 was significantly slower than that of leaves overexpressing of VqWRKY6 alone and overexpressing of VqbZIP1 alone, and the ROS content in the leaf tissues co-overexpressing of VqWRKY6 and VqbZIP1 was significantly higher than that of leaves overexpressing of VqWRKY6 alone and overexpressing of VqbZIP1 alone. In addition, the synergistic regulation of VqWRKY6 and VqbZIP1 could activate PR3 and PR4 of the jasmonate (JA) pathway, and the gene expression levels were significantly upregulated.【Conclusion】 The synergistic effect of VqWRKY6 and VqbZIP1 may improve the disease resistance of grape to powdery mildew by activating the JA pathway, promoting the production of ROS, enhancing the expression of disease-resistant genes to inhibit the growth of U. necator. VqWRKY6 and VqbZIP1 are important disease-resistant genetic resources, and ‘Shang-24’ is an important disease-resistant germplasm resource.

Synthesis and Bioactivity of Sex Pheromone Analogues of Trachea atriplicis

REN ZiQi, KANG YuJie, LI HaiZhen, WANG LianGang, MA HaoYun, LI Hui, WANG LiuYang, MEI XiangDong, NING Jun

Scientia Agricultura Sinica. 2022, 55(23): 4640-4650. doi:10.3864/j.issn.0578-1752.2022.23.006

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【Background】 Trachea atriplicis is an important leaf-eating pest, which has caused great harm to agriculture and forestry in North China, East China, and other regions in recent years due to its increasing numbers and the level of hazards, especially posing a serious threat to the yield of crops such as buckwheat.【Objective】Based on the maternal structure (Z)-hexadec-11-en-1-yl acetate (Z11-16:Ac), which is the main component of the sex pheromone of T. atriplicis, six sex pheromone analogues with different bioactivities were synthesized. The aim was to open up a novel environment-friendly control strategy for the comprehensive treatment of T. atriplicis.【Method】 As the raw material, the sex pheromone precursor (Z)-hexadec-11-en-1-ol (Z11-16:OH) was used to react with an acid anhydride or acyl chloride under the action of an acid binding agent and catalyst. After being separated and purified by silica gel column chromatography, a total of six synthetic pheromone analogues were obtained, including M3 and M4, the sex pheromone analogues with carbamate polar groups, M5 and M6, the sex pheromone analogues with the unsaturated bond at the end of the polar group, and sex pheromone analogues M7 and M8 with halogen atom modification. The electrophysiological response values under direct stimulation of different doses with sex pheromone analogues were measured by the EAG test, and the biological activities of sex pheromone analogues were verified in field experiments conducted in Yanqing, Beijing in 2021.【Result】 The EAG test showed that analogues M1, M2, M6, M7, and M8 had significant dose-dependent relationships. In particular, T. atriplicis showed the strongest response to analogue M7, with a response value of 2.36 mV at the dose of 100 μg, which was 77.9% of that of the response value of Z11-16:Ac at the same dose. While there were significant differences (P<0.05) between the relative response values of M3-M5 and sex pheromone components Z11-16:Ac at the doses of 10-1 000 μg. The M5 displayed lowest EAG response and the highest relative response value was only 21.2% of the sex pheromone fraction. As shown in the field trials, the average capture amount of analogues M1 and M5 was 38.00 and 35.67 moths/trap/15 d at an addition of 1 μg, respectively, which was significantly different compared with the sex pheromone control. The analogue M7 trapped 29.67 moths/trap/15 d at an addition of 100 μg, which was also significantly higher than that of the sex pheromone control. The analogue M4 showed potential inhibitory activity against sex pheromone, and the average capture amount decreased gradually with the increase of its addition, the average capture amount was only 3.33 moths/trap/15 d at the dose of 1 000 μg.【Conclusion】 The EAG tests and field trials verified that the analogues M1, M5, and M7 showed significant synergistic activity and could be developed as synergists of sex pheromones. The analogue M4 showed significant inhibitory activity, which could be used as an inhibitor of sex pheromones. This study provides a necessary theoretical basis for the use of sex pheromone analogues to control T. atriplicis in a green way.

Effects of Straw Returning and Potassium Fertilizer on Soil Aggregate and Potassium Distribution Under Rapeseed-Rice Rotation

LIU ShuJun,LI DongChu,HUANG Jing,LIU LiSheng,WU Ding,LI ZhaoQuan,WU YuanFan,ZHANG HuiMin

Scientia Agricultura Sinica. 2022, 55(23): 4651-4663. doi:10.3864/j.issn.0578-1752.2022.23.007

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【Objective】 The study aimed to explore effects of different fertilization on paddy soil aggregate characteristics and potassium distribution under rapeseed-rice rotation and winter fallow by the located experiment test. It was hoped that the study would provide a basis for the sustainable utilization of potassium resources in paddy-upland rotation areas of Southern China. 【Method】 Based on the location experiment of potassium fertilizer in different rotation patterns started in 2016, five treatments were selected, including CK(F) (no fertilization and winter fallow), NPK(F) (NPK fertilizer and winter fallow), NPK(R) (NPK fertilizer and winter rapeseed), NPK (SR+R) (NPK fertilizer with rice straw returning and winter rapeseed), and NP50%K (SR+R) (50% reduction of K fertilizer with rice straw returning and winter rapeseed). The soil samples were collected from the 0-20 cm soil layer of each treatment after rape harvest of the third year for analysis of physical and chemical properties, such as soil aggregates distribution, and exchangeable and non-exchangeable potassium content variation in soil aggregates. The stability of soil aggregates and the distribution of potassium in aggregates were further investigated..【Result】 The soil aggregate fractions of all treatments was the highest in <0.053 mm. Compared with NPK(F), NPK(R) increased the proportion of aggregates of 1-2 mm, 0.5-1 mm and 0.25-0.5 mm by 26.2%-82.6% under the same fertilization treatment. Also the stability of soil aggregates was increased, the proportion of aggregates of >0.25 mm, mean weight diameter (MWD), and geometric mean diameter (GMD) were significantly increased by 30.6%, 31.2% and 82.0%, respectively. Under rapeseed-rice rotation, the proportion of aggregates of >2 mm was increased under NPK(SR+R), which was 69.7% higher than that under NPK(R). The exchangeable potassium content in soil aggregates decreased with decreasing particle size under all treatments. NPK(SR+R) significantly increased exchangeable potassium content in all aggregate fractions by 22.2%-46.0% compared with NPK(R) under rapeseed-rice rotation. NP50%K(SR+R) significantly reduced the exchangeable potassium content in aggregates of >0.5 mm by 19.4%-20.6% than NPK(SR+R). Compared with NPK(F), three fertilization treatments under rapeseed-rice rotation all reduced the non-exchangeable potassium content in aggregate fractions. Under all treatments, the contribution rate of different fractions of soil aggregates to the bulk soil in potassium was the highest in <0.053 mm. Compared with NPK(F), NPK(R) significantly increased the contribution rates of 1-2 mm and 0.5-1 mm aggregates to bulk soil in potassium by 82.6%, 52.1% (exchangeable potassium ) and 105.5%, 36.9% (non-exchangeable potassium), respectively.【Conclusion】 The rapeseed-rice rotation could increase MWD, GMD, macroaggregate proportion and the contribution rate of macroaggregate to the bulk soil in potassium and improve soil structure. Under this rotation pattern, the chemical fertilizer combined with rice straw returning could increase the exchangeable potassium content in all aggregate fractions, which improved the potassium supply in paddy soil. However, the rapeseed-rice rotation reduced the content of non-exchangeable potassium in soil aggregates due to high potassium demand, and the input of potassium fertilizer should be appropriately increased.

Carbon and Nitrogen Fixation Characteristics of Maize Root and Straw Residues in Brown Soil Under High and Low Fertility

LI JiaYan,SUN LiangJie,MA Nan,WANG Feng,WANG JingKuan

Scientia Agricultura Sinica. 2022, 55(23): 4664-4677. doi:10.3864/j.issn.0578-1752.2022.23.008

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【Objective】 Straw returning to the field is one of important measures to increase soil carbon (C) fixation and to improve soil nutrient status. Studying the C and nitrogen (N) fixation characteristics of different parts of maize residues in the soil is of great significance for clarifying the C and N sequestration mechanism under straw returning. 【Method】 This study was based on the long-term experiment station of Shenyang Agricultural University. The soils treated with no fertilization and organic fertilizer combined with chemical fertilizers were collected as low fertility (LF) and high fertility (HF) soils, respectively, and combined with ¹³C and ¹⁵N double-labeled maize residues. Maize stalk and leaf part (S) as well as root part (R) are mixed with collected soils, respectively, and incubated at 25℃. In the experiment, the samples were taken on the 1^st, 30^th, 60^th, 180^th and 360^th days to determine the total organic C (SOC), total N (TN) content, and their isotopic abundances, and to analyze C and N fixing dynamics of different parts of maize residues in the soil with different fertility levels..【Result】 Adding maize residues significantly increased soil SOC, and could still increase by 14.0% after one year. After adding maize residues, a small part of N in the soil system might be lost by denitrification, and both added N deriving from maize residues and native N in soil were contributed in the lost. Compared with the addition of root residues, the addition of straw residues was more conducive to retaining the added residual C and N, which had a stronger effect on stimulating the local C decomposition and N loss. In contrast, the root residues tended to be decomposed, while the native soil C and N were relatively protected. Although the fixation of added residual C in low-fertility soil was higher than that in high-fertility soil, the fixed added residual C presented a greater contribution to the promotion of C pool in the low-fertility soil. Under the treatment of residues addition, C/N and ¹³C/¹⁵N (representing residue-deriving C/N in the soil) in low-fertility soil were significantly higher than those in high-fertility soil. However, the results showed that ¹³C/¹⁵N was not the main factor limiting the decomposition and fixation of residues in low-fertility soil, which might be related to the distinctive soil microbial community favoring of utilizing the specific substrate under the long-term substrate selection..【Conclusion】 Adding maize residues from different parts of the soil could significantly increase soil carbon and nitrogen levels, but the immobilization strategies of new carbon/nitrogen in the residues and soil old carbon/nitrogen were different. The low-fertility soil had higher capacity to fix exogenous carbon and less fertility, and their fixation of residues in different parts was not affected by the limitation of C/N from exogenous residues in this study.

Net Anthropogenic Nitrogen Inputs and Its Influencing Factors in Three Typical Watersheds of China

ZHANG TianPeng,YAN TieZhu,JIN PingZhong,LEI QiuLiang,LIAN HuiShu,LI Ying,LI XiaoHong,OU HuiPing,ZHOU JiaoGen,DU XinZhong,WU ShuXia,LIU HongBin

Scientia Agricultura Sinica. 2022, 55(23): 4678-4687. doi:10.3864/j.issn.0578-1752.2022.23.009

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【Objective】 Net anthropogenic nitrogen input (NANI) is one of the important causes to non-point source pollution. In order to investigate the spatio-temporal characteristics and influencing factors of net anthropogenic nitrogen input, the key source areas were identified and the key sources in watersheds were analyzed, so as to provide data support for solving the problem of non-point source nitrogen pollution. 【Method】 Three watersheds were selected according to their characteristics, among which Xiangxi River watershed was a typical agriculture watershed, Erhai watershed was comprehensively promotion model of green agricultural development, and Taihu watershed was a typical urban centralized watershed. Based on the NANI model, the data was obtained through statistical yearbook and literature review to evaluate NANI of the three typical watersheds.【Result】 In terms of NANI intensity, the average value of NANI in the three typical watersheds was ranked as follows: Taihu watershed (13 241 kg·km^-2·a^-1), Xiangxi River watershed (2 183 kg·km^-2·a^-1), and Erhai watershed (1 582 kg·km^-2·a^-1). In terms of NANI sources, nitrogen application (Nfer) and food/feed nitrogen (Nim) were the largest sources of NANI, accounting for 58%-97%. The NANI contribution ranked in the order of nitrogen application, food/feed nitrogen input, nitrogen deposition input, and crop nitrogen fixation input. In terms of time, food/feed nitrogen input of Xiangxi River watershed decreased by 23 percentage points from 2010 to 2019, while nitrogen deposition increased by 34 percentage points. From 2010 to 2019, nitrogen application in NANI decreased by 86 percentage points in Erhai watershed. From 2010 to 2019, the input of food/feed nitrogen to NANI in Taihu watershed increased by 31 percentage points, while the input of crop nitrogen fixation and nitrogen deposition decreased by 14 and 12 percentage points, respectively. In terms of influencing factors, NANI was significantly correlated with urban population density in the three typical watersheds (P<0.05), and NANI increased with the increase of urban population density. The Xiangxi River watershed had a significant effect on the proportion of cultivated land and NANI fitting (P<0.05), but the Erhai watershed and Taihu watershed was not significant effect (P>0.05)..【Conclusion】 Zhaojun town, Xiakou town and Huangliang town in Xiangxi River watershed, Xiaguan town, Shangguan town and Fengyi town in Erhai watershed, and Zhangjiagang City, Xiucheng District in Jiaxing City, Gongshu District and Nanhui District in Taihu watershed were the key source areas of NANI. Fertilizer application was the main source of NANI in Xiangxi River watershed where is mainly agricultural. The input of food/feed nitrogen and fertilizer nitrogen were the main sources of NANI in Taihu watershed where is mainly urbanization. The green agricultural development model could significantly reduce net anthropogenic nitrogen input. Therefore, it was beneficial to control agricultural non-point source pollution by vigorously promoting agricultural green development measures and effectively reducing the input of feed and fertilizer in key source areas.

Effects of Artificial Fruit Thinning on Sugar and Acid Content and Expression of Metabolism-Related Genes in Fruit of Beni-Madonna Tangor

SONG JiangTao,SHEN DanDan,GONG XuChen,SHANG XiangMing,LI ChunLong,CAI YongXi,YUE JianPing,WANG ShuaiLing,ZHANG PuFen,XIE ZongZhou,LIU JiHong

Scientia Agricultura Sinica. 2022, 55(23): 4688-4701. doi:10.3864/j.issn.0578-1752.2022.23.010

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【Background】 Beni-Madonna tangor (Citrus nanko × C. amakusa), a hybrid cultivar of citrus, is favored by consumers because of its excellent quality. Fruit thinning is a technique commonly used to improve fruit quality in the process of agricultural production. It can not only significantly increase the fruit volume, but also increase the content of soluble solids in the fruit. However, the specific mechanism of fruit thinning to increase the content of soluble solids in the fruit of Beni-Madonna is still unclear. 【Objective】 In this study, four-year-old Beni-Madonna tangor plants, with trifoliate orange (Poncirus trifoliata) as a base rootstock and Ponkan as an intermediate stock, were used as experimental materials to explore the specific mechanism of fruit thinning on affecting the change of sugar and acid content in fruit. 【Method】Fruit thinning was carried out at the young fruit stage of Beni-Madonna fruit. The horizontal and vertical stems of fruit thinning and non-fruit thinning were measured every half a month or so. The samples were taken back and the single fruit weight and sugar and acid content were measured. The activities of sugar and acid metabolism related enzymes and the relative expression of their corresponding coding genes were measured when the sugar and acid content were significantly different..【Result】 The fruit thinning significantly increased the horizontal and vertical stems and single fruit weight of Beni-Madonna fruit in the middle and late stages of fruit growth, significantly accelerated the degradation rate of citrate content, but did not affect the final citrate content at fruit maturity, significantly increased the fruit glucose and sucrose content, but had no significant effect on fructose content. The fruit thinning significantly increased the activities of sucrose synthase SSⅡ (synthesis direction), sucrose synthase SSⅠ (decomposition direction), sucrose phosphate synthase (SPS) in sucrose metabolism and cytosolic aconitase (Cyt-ACO), cytoplasmic isocitrate dehydrogenase (ICDH) and glutamine synthetase (GS) implicated in citric acid catabolism. In addition, fruit thinning significantly promoted the early relative expression of CitSS3 and the relative expression of CitSPSs at all stages in the process of sucrose metabolism, and the relative expression of CitACO3, CitNADP-IDH1, CitACLα1/β, CitGAD1 and CitGSs in the process of citric acid metabolism in the early and middle stages..【Conclusion】 The artificial fruit thinning mainly promoted the accumulation of soluble sugar in fruit by enhancing the enzyme activity of sucrose phosphate synthase in the process of sucrose anabolism, improved the activity and gene expression level of related enzymes in the process of citric acid catabolism, and accelerated citric acid degradation, thus playing an important role in improving fruit quality.

Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage

GUO ShaoLei, XU JianLan, WANG XiaoJun, SU ZiWen, ZHANG BinBin, MA RuiJuan, YU MingLiang

Scientia Agricultura Sinica. 2022, 55(23): 4702-4716. doi:10.3864/j.issn.0578-1752.2022.23.011

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【Objective】 The aim of this study was to identify members of the XTH gene family from peach, and to analyze the expression of PpXTHs in peach fruit with different textures during storage, which not only provided data for the research on the candidate PpXTHs involved in peach fruit softening, but also laid the foundation for further study on the PpXTHs function in peach fruit softening.【Method】 The HMM profiles of the Glyco_hydro_16 domain and XET_C domain were used to search all XTH proteins with the Hmmer 3.1 software in the peach protein database. The molecular weight, theoretical isoelectric point and other physicochemical properties were then predicted by the online tool ProtParam. PpXTHs subcellular localization were predicted by the online software Plant-mPLoc. The MEGA 11 software was used to construct a phylogenetic tree. The online tool MEME was used to analyze conserved motifs, the conserved motifs, conserved protein domains and gene structure maps were draw by Tbtools. According to the PpXTH gene family location information, chromosome mapping was carried out with MapChart software. The expressions of PpXTHs in peach fruit with different textures during storage were monitored by qRT-PCR..【Result】 A total of 27 PpXTH genes were systematically identified from peach, which were distributed on seven chromosomes. Based on the phylogenetic tree, the ancestral group, Ⅰ/Ⅱ subfamily, ⅢA subfamily and ⅢB subfamily were classified. In addition, according to the analysis of protein domains, all PpXTH gene family proteins had Glyco_hydro_16 and XET_C conserved domain. The results from qRT-PCR analysis showed that PpXTH33 belonging to the ⅢB subfamily was upregulated as the storage period increased in melting peach fruit, with the expression being markedly higher than that during the storage period of stony hard peach fruit. The positive clone sequencing was consistent with the coding sequence of the Prunus persica reference genome with a length of 924 bp for a 307 amino acid sequence. The PpXTH33 combined with green fluorescent protein may mainly located in the cell wall and nucleus detected by confocal laser scanning microscopy..【Conclusion】 All 27 PpXTHs protein structures contained two highly XTH conserved domains and the genes were distributed on seven chromosomes. The expression characteristics of PpXTH33 in peach fruit with different flesh textures during storage suggested that PpXTH33 was closely associated with peach fruit softening during storage.

Effects of Low Oxygen/High Carbon Dioxide Controlled Atmosphere Combined with 1-Methylcyclopropene on Quality of Yuluxiang Pear During Cold Storage

JIA XiaoHui, ZHANG XinNan, LIU BaiLin, MA FengLi, DU YanMin, WANG WenHui

Scientia Agricultura Sinica. 2022, 55(23): 4717-4727. doi:10.3864/j.issn.0578-1752.2022.23.012

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【Objective】 The objective of this study was to clarify the effects of a controlled atmosphere (CA) with low oxygen (O₂)/high carbon dioxide (CO₂) on chlorophyll maintenance and quality of Yuluxiang pears during cold storage, so as to provide a theoretical basis and technical support for prolonging the storage life of Yuluxiang pear.【Method】 The storage experiments were performed with commercial mature Yuluxiang pears treated with 1.0 μL·L^-11-Methylcyclopropene (1-MCP), 1% O₂, 3% CO₂, and 1.0 μL·L^-11-MCP combined with 1% O₂ and 3% CO₂, with air treatment as the control. The peel color, chlorophyll content, firmness, total soluble solids, titratable acid, ascorbic acid, and other quality indices were measured at 210 and 240 days of storage and 7 days of shelf life, respectively. Ethanol and acetaldehyde content, ethylene production, and respiratory rate of the fruits were detected by gas chromatography, and the browning indices of the fruit stalk and core were investigated and calculated.【Result】 Compared with ordinary cold storage, 1-MCP, CA, and CA+1-MCP could better maintain the green color of Yuluxiang pear fruits and effectively reduce the degree of greasiness on the fruit surface. CA+1-MCP had a more pronounced effect on the green color maintenance and greasiness control of the peel when they were refrigerated for 240 days and 240+7 days. 1-MCP and CA could inhibit the decline in fruit firmness, total soluble solids, and titratable acid. CA could inhibit browning of the core and stalk, but CA used alone reduced the content of ascorbic acid in the fruits, and CA+1-MCP slowed the decline of ascorbic acid in CA fruits. The inhibitory effect of CA+1-MCP on ethanol and acetaldehyde was more apparent when stored for 240 days, and the ethanol content of 20 mg·L^-1was below the tolerance threshold of Yuluxiang pears. CA+1-MCP and 1-MCP had strong inhibitory effects on ethylene production across the entire storage period. At 240 days, CA+1-MCP and CA had a stronger inhibitory effect on the respiratory rate than 1-MCP.【Conclusion】 Yuluxiang pears were respiratory climacteric pears, resistant to low O₂ and high CO₂. The freshness maintenance effect of CA+1-MCP on Yuluxiang pear was maintained after 210 days. Therefore, when the cold storage period was less than 210 days, a single 1-MCP treatment of 1.0 μL·L^-1 could be used. When the cold storage period was greater than 210 days, the fruit should be first treated with 1.0 μL·L^-1 1-MCP, and then stored under CA with low O₂/high CO₂ of 1% O₂ and 3% CO₂, which could maintain the appearance and internal quality of fruits, as well as significantly prolong the cold storage life.

Physiological Metabolic Rol e of Nanocomposite Packaged Agaricus bisporus During Postharvest Cold Storage Analyzed by TMT-Based Quantitative Proteomics

WANG Chao,FANG DongLu,ZHANG PanRong,JIANG Wen,PEI Fei,HU QiuHui,MA Ning

Scientia Agricultura Sinica. 2022, 55(23): 4728-4742. doi:10.3864/j.issn.0578-1752.2022.23.013

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【Background】 Agaricus bisporus (A. bisporus) is prone to quality deterioration, such as umbrella opening, water loss, and browning after harvest, which seriously affects the storage quality and commercial value. Our previous research has confirmed that the nanocomposite packaging material (Nano-PM) could effectively delay the postharvest quality deterioration of A. bisporus, but the preservation mechanism is still unclear. 【Objective】 In this study, the differentially expressed proteins of A. bisporus in Nano-PM and polyethylene packaging material (Normal-PM) during storage were analyzed by Tandem Mass Tag (TMT) quantitative proteomics technology. The preservation mechanism of Nano-PM on A. bisporus was further explored. 【Method】 A. bisporus was taken as the research object. The Nano-PM was used for the preservation of A. bisporus, and the Normal-PM was used as the control. The protein extraction and trypsin hydrolysis were performed on A. bisporus during storage. The differentially expressed proteins were screened by TMT labeling and liquid chromatography-tandem mass spectrometry detection. Combined with bioinformatics analysis, the main metabolic pathways involved in differential proteins were studied. Quantitative real-time polymerase chain reaction (qPCR) technology was used to determine the gene expression levels of differential proteins..【Result】 The Nano-PM effectively maintained the appearance quality of A. bisporus and delayed the increase of cell membrane permeability. The number of differential proteins in two groups increased during storage. In the middle (6 d) and late (10 d) stages of storage, the numbers of differential proteins were 62 and 148, respectively. Among them, 22 differential proteins were common. Combined with bioinformatics analysis, these differential proteins were mainly related to pathways, such as energy metabolism and lipid metabolism. The lipid metabolism pathway was mainly analyzed, and the results showed that the Nano-PM had a regulatory effect on the membrane lipid metabolism of A. bisporus. Compared with the Normal-PM, the protein expression of fatty acid synthase, phosphorylcholine cytidylyltransferase, and phosphatidic acid phosphatase under Nano-PM were up-regulated, while the protein expression of key enzymes in membrane lipid degradation, such as phospholipase D and lipase, were down-regulated. At the gene level, the expression of genes encoding these proteins were consistent with the proteomics results..【Conclusion】 The differential proteins of different packaged A. bisporus during storage could be screened and analyzed by TMT-based quantitative proteomics technology. Nano-PM regulated the membrane lipid metabolism of A. bisporus, and inhibited the expression of membrane lipid degradation-related enzymes, which effectively delayed the increase in cell membrane permeability of A. bisporus, maintained the structure and function of the cell membrane, and delayed the quality deterioration of A. bisporus during storage.

Proteomic Analysis of Sperm with Different Freezing Tolerance in Erhualian Boar

TONG ShiFeng,REN ZhiBin,LIN Fei,GE YuZhu,TAO JingLi,LIU Yang

Scientia Agricultura Sinica. 2022, 55(23): 4743-4752. doi:10.3864/j.issn.0578-1752.2022.23.014

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【Objective】 Erhualian pig is one of the excellent indigenous pig breeds in China. Frozen semen can improve the utilization rate of Erhualian boar and strengthen the protection of germplasm resources. However, the frozen semen of Erhualian pigs can not meet the production demand. This study analyzed the proteomics of Erhualian boar sperm with different freezing tolerance to promote the screening of protein markers of Erhualian boar sperm freezing tolerance, analyze the influencing factors of sperm freezing tolerance from the genetic level, and provide reference basis for improving sperm freezing tolerance. 【Method】 In this study, semen from14 Erhualian boars were frozen and their quality was analyzed. Boars with good and poor freezability ejaculates (GFE and PFE, n=3) were selected respectively, according to the motility and progressive motility of frozen-thawed spermatozoa. Using tandem mass tag (TMT) analyzed the sperm proteomics of GFE and PFE, and the differentially abundant proteins (DAPs) of spermatozoa of GFE and PFE were identified. The functions of DAPs were annotated by GO enrichment analysis, and the biological pathways of DAPs were annotated by KEGG enrichment analysis. The interaction network of DAPs was analyzed by STRING protein interaction database, and the protein-protein interaction (PPI) network was constructed by Cytoscape software..【Result】 The study found that there were 138 DAPs between GFE and PFE. Compared with GFE, 109 DAPs were up-regulated and 29 DAPs were down-regulated in PFE. GO enrichment analysis showed that a total of 124 DAPs were enriched to 47 GO terms, mainly enriched in “cellular process” “single-organism process” “binding” “cell” “cell part” and so on. KEGG enrichment analysis showed that DAPs were mainly enriched in “Renin-angiotensin system” “Complement and coagulation cascades” “Platelet activation” and so on. There were 53 nodes and 69 edges in the PPI network, among which 8 proteins showed high combine_score, including ALB, ACRBP, ACR, ZAN, ZPBP2, HSPA5, FGB and FGG. The functions of these proteins were mainly enriched in GO terms closely related to animal reproductive physiology and spermatogenesis, such as “single fertilization” “sexual reproduction” and “spermatid development”. Further analysis shows that DAPs in GFE and PFE mainly included redox related proteins (GSTM3, PRDX5, ALB, PDIA1, PDIA4), spermatogenesis and sperm function related proteins (HSPA5, MFGE8, DNAH1, DNAH7, CUL3), energy related proteins (HK1, PGM1), apoptosis and inflammation related proteins (THBS1, ELSPBP1, CP), and PPI network analysis shows that three DAPs (MME, ANPEP and PRCP) interact in renin-angiotensin system (RAS)..【Conclusion】 There are significant differences in sperm protein components of Erhualian boar with different freezing tolerance, which may affect the freezing performance of sperm. These DAPs can be used as candidate protein markers of sperm freezing tolerance.

Screening of Key Regulatory Genes for Litter Size Trait Based on Whole Genome Re-Sequencing in Goats (Capra hircus)

LI Heng,ZI XiangDong,WANG Hui,XIONG Yan,LÜ MingJie,LIU Yu,JIANG XuDong

Scientia Agricultura Sinica. 2022, 55(23): 4753-4768. doi:10.3864/j.issn.0578-1752.2022.23.015

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【Objective】 The purpose of this study was to analyze the genome of different fecundity populations of goats (Capra hircus) and to explore the key regulatory genes involved in the regulation of litter size traits of Chuanzhong black goats (CBGs), and to provide the theoretical reference for analyzing the genetic mechanism of litter size traits and molecular genetic improvement of fecundity in goats. 【Method】 The high fecundity (HF) CBG does (n = 6) that produced 4-6 kids per doe kidding and low fecundity (LF) does (n = 6) that produced only one kid per doe kidding were chosen in this study. The jugular blood samples were collected to extract genomic DNA. The 350 bp double-terminal sequencing library was constructed, and then 12 whole genome libraries were resequenced by IlluminaHiSeqPE150 platform. The clean data from sequencing were mapped to goat reference genome ARS1 by using BWA software, and two whole-genome scanning analysis methods (Fst and Hp) were used to comprehensively analyze the high-quality SNPs obtained to identify candidate regions. GO analysis and KEGG pathway analysis were performed on the G:Profiler and KOBAS online databases, respectively, to screen candidate genes for regulating the number of kids in CBGs. To further identify the key genetic markers that regulate the number of kids, the synonymous and non-synonymous single nucleotide polymorphisms (SNPs) of reproductive candidate genes were mapped and screened according to the variation analysis report of genome resequencing. The amplified products of 12 goat samples were sequenced by Sanger sequencing to verify the resequencing results.【Result】 A total of 431.50 Gb clean data were obtained from the genome resequencing study of 12 CBGs. Through mutation detection and annotation, 7 771 417 SNPs were detected in HF group and 8 935 907 SNPs were detected in LF group, and all types of the LF group SNPs were more than those in HF group. The windows that reach the maximum ZFst value of top 5% and the minimum ZHp value of top 5% were set as candidate regions. A total of 130 strong selection signals were annotated in the regions with low heterozygosity and high genetic differentiation, of which 84, 59 and 13 genes were annotated in HF group, LF group and shared window, respectively. GO enrichment analysis and KEGG pathway showed that 19 candidate genes were involved in the regulation of reproduction, reproduction and embryonic development of CBG, including 11 HF group-specific candidate genes (ADCY10, DRD1, HS6ST1, IGFBP7, MSX2, NOG, NPHP4, PAPPA, PRLHR, TDRP, and XYLT1), and five strong selection signal genes (ANXA5, IGF1, EDNRA, FANCL, and TAC1) in LF group, and three window genes (AKR1B3, HDAC4 and OPRM1) in HF group shared with LF group. The most GO terms, such as G-protein-coupled receptor activity, hormone response and neuropeptide signal pathway, contained these 19 candidate genes. In addition, nine of the 14 HF candidate genes were significantly enriched in metabolic pathway, neuroactive ligand-receptor interaction, glycosaminoglycan-heparan sulfate/heparin biosynthesis, calcium signal pathway, cAMP signal pathway and folate biosynthesis KEGG pathways (P<0.05). Among the 19 reproductive candidate genes, there were two synonymous mutations (MSX2 G771T, ADCY10 A4662G) and two non-synonymous mutations (PRLHR G529, DRD1 A281T), which were only located in the HF candidate genes. The Sanger sequencing showed that polymorphisms of MSX2, PRLHR and DRD1 gene mutations could be detected, and this result was consistent with the results of genome resequencing, in which PRLHR G529A polymorphism led to alanine mutation to threonine, and DRD1 A281T polymorphism led to early termination of translation.【Conclusion】 A total of 11 HF group-specific candidate genes were found in this study, which were speculated to be the key regulatory genes for fecundity trait. The mutations of PRLHR gene exon G529A and DRD1 exon A281T might be the key genetic markers for regulating prolificacy traits in goats, which had great application value in improving reproductive performance of goats.

Screening of Wnt3a SNPs and Its Association Analysis with Skin Feather Follicle Density Traits in Chicken

TU YunJie,JI GaiGe,ZHANG Ming,LIU YiFan,JU XiaoJun,SHAN YanJu,ZOU JianMin,LI Hua,CHEN ZhiWu,SHU JingTing

Scientia Agricultura Sinica. 2022, 55(23): 4769-4780. doi:10.3864/j.issn.0578-1752.2022.23.016

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【Objective】 The Wnt signaling pathway plays an important role in the development of animal skin feather follicles. The results of previous studies indicated that the Wnt3a might be an important candidate gene that had effects on the chicken feather follicle density. In order to further verify the role of Wnt3a in the growth and development of skin feather follicle density, Wnt3a SNPs were screened and their association with feather follicle density would be analyzed in Jinling Hua chicken. The study could provide a reference for “breeding by molecular writing” of slaughter-type broilers with beautiful carcasses.【Method】 The SNPs of Wnt3a gene were screened by PCR amplification and direct sequencing, and the correlation between a single SNP marker and skin feather follicle density traits was analyzed. Haploview software was used to analyze the degree of linkage disequilibrium (LD) of these SNP loci, and the correlation between different haplotype combinations and feather follicle density traits was also analyzed..【Result】 A total of 14 SNP sites were found, and one SNP site (SNP1) was found in the second exon, which was a synonymous mutation. Four mutation sites (SNP2-SNP5) were found in the second intron, and 9 SNP sites (SNP6-SNP14) were found in the third intron. The chi-square test showed that one mutation site (SNP1) in the second exon and three mutation sites (SNP3-SNP5) in the second intron were all in the Hardy-Weinberg equilibrium (P>0.05), 9 mutation sites (SNP6-SNP14) of the third intron deviated from Hardy-Weinberg equilibrium (P<0.05). The expected heterozygosity (He) of SNP1-SNP5 was less than 0.50, and the polymorphic information content (PIC) was less than 0.25. The genetic polymorphisms of these 5 SNP loci were low. The third intron had 9 mutation sites, PIC of SNP6, SNP7, SNP9 sites was less than 0.25, and the other 6 mutation sites were 0.25<PIC<0.5, which were moderately polymorphic. Single-marker association analysis showed that the number of skin feather follicle with SNP2 locus of AG genotype in males and females was significantly higher than that of GG genotype (P<0.05). The number of skin feather follicles with SNP8 locus of AA and GG genotypes in females was significantly higher than that of the AG genotype (P<0.05). The skin feather follicles density in the three genotypes in males was not significantly different. The linkage disequilibrium analysis of 14 SNPs showed that SNP6-SNP13 and SNP3-SNP5 had a strong linkage disequilibrium, respectively. SNP3, SNP4, and SNP5 produced three haplotype combinations after the combination of the two haplotypes linked by SNP3- SNP5. Association analysis found that the skin feather follicle density of the three haplotype combinations in males and females were not significantly different. After the combination of 5 main haplotypes at SNP6-SNP13 locus, the males had 7 haplotype combinations, and the skin feather follicles density was not significant. Females had 8 haplotype combinations, of which H1H1 (AACCAATTTTAATTCC) had the highest skin feather follicles density. SNP2 and SNP8 were significantly correlated with skin feather follicle density, and the haplotype combination H1H1(AGAA) and H1H2 (AGAG) were the dominant haplotypes in hens.【Conclusion】 Fourteen SNPs of Wnt3a were screened. Among them, individuals with different genotypes at rs2587721 G>A (SNP2) and rs2555967G>A (SNP8) locus had significant differences in feather follicle density. Eight SNPs (SNP6-SNP13) loci were in strong linkage disequilibrium, and the combination of H1H1 had the highest feather follicles density in females. The haplotype combination of SNP2 and SNP8 of Wnt3a H1H (AGAA), H1H2 (AGAG) and SNP6-SNP13 linked to produce the H1H1 (AACCAATTTTAATTCC) haplotype combination were significantly correlated with feather follicle density in females, which provided important genetic information for “breeding by molecular writing” on chicken skin feather follicle density.

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