Scientia Agricultura Sinica

The Effect of indica/Xian Pedigree Introgression in japonica/Geng Rice Breeding in China

XU HAI, LI XIUKUN, LU JIAHAO, JIANG KAI, MA YUE, XU ZHENGJIN, XU QUAN

Scientia Agricultura Sinica. 2023, 56(22): 4359-4370. doi:10.3864/j.issn.0578-1752.2023.22.001

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【Objective】To demonstrate the impact of indica/Xian (XI) pedigree introgression on the yield and quality of japonica/Geng (GJ) rice varieties, providing a theoretical basis and genomic resources for optimizing XI pedigree introgression breeding programs in northern GJ rice.【Method】In this study, the whole genome sequence on Illumina platform was employed to elucidate the effects of XI pedigree introgression on the yield and quality of rice in Northeast China were analyzed using recombinant inbred lines (RIL) derived from the cross between XI and GJ varieties, and 74 major GJ varieties grown from Heilongjiang, Liaoning, Shandong, and Jiangsu provinces as test materials. Using CRISPR/Cas9 gene editing technology to knock out the unfavorable genes introduced by XI pedigree introgression. 【Result】Analysis of RIL revealed a significant positive correlation between XI pedigree introgression and panicle length, grain length, and a negative correlation with head rice ratio. XI pedigree introgression was significantly negatively correlated with Amylose content, and significantly positively correlated with protein content in Jiangsu. With the increase of latitude, the correlation efficiency between XI pedigree introgression and grain shape increased, while the correlation between XI pedigree introgression and panicle length and head rice ratio decreased. The genomic fragments of XI pedigree introgression are unevenly distributed across different chromosomes and are more abundantly present on chromosomes 1, 10, 11, and 12. The XI pedigree introgression of the major cultivars in Jiangsu and Liaoning provinces is significantly higher than that in Heilongjiang and Shandong provinces, and the XI pedigree introgression of the cultivars after 2000 is significantly higher than that before 2000. The XI pedigree introgression includes multiple resistance and fertility-related genes. The project identified an XI pedigree introgression fragment on chromosome 5 of YF47, including the XI type grain regulatory gene GS5 and XI type chalkiness regulatory gene Chalk5, which increased the 1000 grain weight of YF47 but affected its chalkiness-related traits. The project uses CRISPR/Cas9 technology to knock out the Chalk5 gene of YF47. The grain shape of the homozygous gene editing plants is similar to those of YF47, and its chalkiness character has been significantly improved. 【Conclusion】The XI pedigree introgression mainly increases the yield potential of GJ rice by increasing the number of grains per panicle, but has a negative impact on milling quality. Exploring the unfavorable alleles in varieties through high-throughput genome sequencing, combined with CRISPR/Cas9 gene editing, to break the genetic drag in breeding using the cross between XI and GJ, is an efficient breeding strategy that can quickly and accurately improve target traits.

Development and Application of Top-Down Approaches for Estimating Measurement Uncertainty of GMO Quantitative Results

LI Jun, ZHAO Xin, CHEN Hong, LI FeiWu, LIANG JinGang, LI YunJing, WANG HaoQian, GAO HongFei, ZHANG Hua, CHEN ZiYan, WU Gang, SHEN Ping, XU LiQun, WU YuHua

Scientia Agricultura Sinica. 2023, 56(22): 4371-4385. doi:10.3864/j.issn.0578-1752.2023.22.002

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【Objective】The enforcement of labeling regulations on genetically modified organisms (GMOs) requires establishing a standard system for accurate quantification of GMOs that includes the standard or guide for estimating measurement uncertainty (MU). It is urgent to establish a standardized top-down approach for estimating MU of quantitative results, which is conveniently adopted by the general testing laboratories. 【Method】There are two approaches for estimating the MU introduced by precision of quantitative method, one is to establish the equation of MU estimation using data obtained on 15 routine samples based on the "uncertainty function", the other is to evaluate the MU by repeatedly measuring a certified reference material (CRM) and calculating the intermediate precision. The uncertainty introduced by bias is evaluated using a CRM or a sample prepared by laboratory as bias control. The uncertainty of the nominal value of the sample prepared by laboratory is evaluated by using a simplified program based on the preparation process. The MU contributed by method precision and bias are combined into the standard uncertainty of the quantitative results, and then multiplied by the coverage factor k to obtain the expanded uncertainty. 【Result】The event-specific PCR method of genetically modified maize DBN9936 was took as an example. The MU of method precision was evaluated to be 0.76% using simulated DBN9936 routine samples, and to be 0.33% using a CRM (GBW (E) 100901). Compared with routine samples, the MU of method precision evaluated using a CRM is significantly underestimated. The uncertainty introduced by bias was evaluated to be 0.26% using a CRM (GBW (E) 100901) as a bias control. Using a laboratory prepared powder sample and a genomic DNA sample (nominal values of 3.0%) as bias control, the bias uncertainty was evaluated to be 0.20% and 0.19%, respectively. Since the simplified program ignored some uncertainty components, the uncertainty of the nominal value of laboratory prepared samples was estimated to be smaller. By combining the MU of method precision and bias, the expanded uncertainty using routine samples was obtained to be 1.26%, 1.20%, and 1.20%, respectively, the expanded uncertainty using a CRM was 0.84%, 0.78%, and 0.76%, respectively. 【Conclusion】This study established the top-down approaches for MU estimation of quantitative results, testing laboratories should prioritize routine samples to estimate the MU contributed by the method precision, and select CRMs as bias control in principle to evaluate bias uncertainty during GMO quantification.

The Molecular Mechanism of Pod Yield Difference Between Single- Seeding Precision Sowing and Multi-Seeds Sowing of Peanut Based on Transcriptome Analysis

YANG Sha, LIU KeKe, LIU Ying, GUO Feng, WANG JianGuo, GAO HuaXin, MENG JingJing, ZHANG JiaLei, WAN ShuBo

Scientia Agricultura Sinica. 2023, 56(22): 4386-4402. doi:10.3864/j.issn.0578-1752.2023.22.003

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【Objective】In China, in order to ensure the emergence rate and quality of seedlings, the field often adopts multiple seed seeding. However, inter-plant competition in multi-seeds sowing often limits the growth and eventual yield of subsequent plants. In order to solve this contradiction, the team studied and established the high-yield cultivation technology of single-seed precision seeding. The combination of seed saving and yield increase effect of single-seed precision seeding technology can bring greater benefits and realize cost savings and increased efficiency. The differentially expressed genes in peanut leaves, roots and pods under different planting methods were used to explore the regulatory mechanism of single-seeding precision sowing to improve peanut pod yield, providing theoretical basis and technical support for further promoting peanut high yield and high efficiency. 【Method】Peanut variety Huayu 25 was used as the test material, while the yield related indexes of single-seed sowing and multi-seeds sowing were determined. Inverted three leaves, taproot and lateral root of peanut at 30 days after flowering and peanut pod at young fruit stage of chicken head were selected for transcriptome sequencing, and the yield differences of peanut under different sowing methods were revealed on the molecular level. 【Result】Compared with multi-seeds sowing, the pod number per plant, full fruit number per plant, fruit weight per plant and economic coefficient of single-seed sowing were significantly increased. After the transcriptome data is assembled, each library contains an average of 44.3 million readings. Through the analysis of differentially expressed genes, GO and KEGG pathways in different combinations, it was found that the expression levels of transcription factors, photosystem Ⅱ oxygen-releasing complex, chloroplast membrane, oxidation-reduction reaction and other genes involved in the processes of GA signal and light signal transduction were increased in the leaves of plants under single-seed sowing compared with multi-grain cave seeding. Genes related to phenylpropyl metabolism pathway induced by biological and abiotic stress were significantly enriched in roots, including cytochrome P450 gene, oxidation-reduction gene, stress response transcription factor and signal regulatory protein. The accumulation of starch and sucrose metabolism genes was more conducive to seed kernel enrichment during pod development. 【Conclusion】The up-regulated expression of photosynthetic related genes in peanut leaves at seedling stage could promote the increase of photosynthetic efficiency, which was closely related to the increase of yield. Single-seed sowing improved the ability of root system to resist biological and abiotic stress, and combined with the up-regulation of energy and material related genes in the early stage of pod development, it was beneficial for the development of underground peanut pod and increased peanut yield.

Prediction of Water Content of Winter Wheat Plant Based on Comprehensive Index Synergetic Optimization

GAO ChenKai, LIU ShuiMiao, LI YuMing, WU PengNian, WANG YanLi, LIU ChangShuo, QIAO YiBo, GUAN XiaoKang, WANG TongChao, WEN PengFei

Scientia Agricultura Sinica. 2023, 56(22): 4403-4416. doi:10.3864/j.issn.0578-1752.2023.22.004

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【Objective】To find a more comprehensive and accurate method to monitor the water deficit and to provide a theoretical basis for drought relief of winter wheat, the present study was conducted to construct an inversion model of plant water content (PWC) at different growth stages based on three comprehensive indexes, namely, canopy temperature, morphology and physiology indexes of winter wheat.【Method】The winter wheat was studied by setting up three water treatments (water deficit treatment W1: 35 mm, water deficit treatment W2: 48 mm, and control treatment W3: 68 mm) and two wheat varieties (general drought resistant variety Luomai 22 and weak drought resistant variety Zhoumai 27). Canopy temperature parameters (canopy temperature standard deviation (CTSD) and crop water stress index (CWSI)), morphological indicators (plant height, stem diameter, aboveground biomass, and leaf aera index (LAI)) and physiological indicators (stomatal conductance, transpiration rate, and photosynthetic rate) of winter wheat were obtained at jointing, booting, and filling stages, respectively. Comprehensive temperature parameter indicators (CTPI), comprehensive growth indicators (CGI) and comprehensive physiological indicators (CPI) based on the average weight principle were constructed. The correlation between PWC and comprehensive indicators was analyzed, and multiple linear regression (MLR), partial least squares recurrence (PLSR) and support vector machine (SVM) methods were used to construct the PWC inversion model based on comprehensive indicators according to the growth period.【Result】The canopy temperature parameters, morphology and physiological indexes of winter wheat at different growth stages showed significant differences between water deficit treatments (W1, W2) and control treatment (W3) (P<0.05). Comprehensive indicators (CTPI, CGI and CPI) at booting and filling stages have a significant correlation with PWC, with correlation coefficients (r) of -0.70 (-0.78), 0.84 (0.80) and 0.83 (0.76), respectively. Using MLR, PLSR and SVM methods, the PWC inversion prediction model based on comprehensive indicators (CTPI, CGI and CPI) has high prediction accuracy, among which the PWC model built by SVM is the best, R²_cal (R²_val), RMSE_cal (RMSE_val), and nRMSE_cal (nRMSE_val) were 0.878 (0.815), 2.06% (2.37%), and 3.10% (3.33%), respectively.【Conclusion】The SVM-PWC model based on the comprehensive indicators CTPI, CGI and CPI can well predict the water deficit of winter wheat at different growth stages, and provide theoretical basis for drought prevention and drought resistance of winter wheat in the Huang-Huai-Hai Plain.

Effect of Light Intensity on Leaf Hydraulic Conductivity and Vein Traits of Soybean at Seedling Stage

GAO Jing, CHEN JiYu, TAN XianMing, WU YuShan, YANG WenYu, YANG Feng

Scientia Agricultura Sinica. 2023, 56(22): 4417-4427. doi:10.3864/j.issn.0578-1752.2023.22.005

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【Objective】The objective of this study is to explore the effects of light intensity on leaf hydraulic conductivity, photosynthetic traits, and water potential in soybean seedlings, analyze the adaptive mechanisms of leaf vein traits in response to varying light intensities, and to provide theoretical support for enhancing future light energy utilization in soybean.【Method】Two soybean varieties, Nandou 12 (shade-tolerant) and Guixia 7 (shade-sensitive), were cultivated and placed in growth chambers. The plants were exposed to varying light conditions, including high light intensity (HL) at (424.47±12.32) µmol·m^-²·s^-¹, medium light intensity (ML) at (162.52±20.31) µmol·m^-²·s^-¹, and low light intensity (LL) at (93.93±9.87) µmol·m^-²·s^-¹. After a 20-day treatment period, the impacts of different light intensities on hydraulic conductivity, photosynthetic parameters, leaf water potential, and leaf vein traits in the seedling leaves of soybean were examined.【Result】Compared with HL treatment, the leaf hydraulic conductivity of Nandou 12 and Guixia 7 under LL treatment was significantly decreased, and the leaf hydraulic conductivity of Nandou 12 under the three treatments was significantly higher than that of Guixia 7 under the three treatments. Compared with HL treatment, the leaf hydraulic conductivity of Nandu 12 under ML and LL treatments decreased by 7.56% and 21.24%, stomatal conductance decreased by 43.96% and 58.89%, and net photosynthetic rate decreased by 29.44% and 46.49%, respectively. Similarly, the leaf hydraulic conductivity of Guixia 7 under the ML and LL treatments decreased by 42.16% and 23.71%, stomatal conductance decreased by 54.55% and 45.79%, and net photosynthetic rate decreased by 37.03% and 42.06%, respectively. Additionally, no statistically significant differences were observed in the leaf water potential of both soybean varieties across the various treatments. Notably, leaf hydraulic conductivity and stomatal conductance of soybean exhibited a highly significant positive correlation (P<0.01) under the three light intensity treatments. As the light intensity decreased, a positive correlation was observed between leaf hydraulic conductivity and net photosynthetic rate (P<0.05) as well as stomatal conductance (P<0.01). Conversely, there was a noticeable decrease in the minor leaf vein density and the area of xylem conduits in major and minor veins under the ML and LL treatments for both soybean varieties. In the case of the minor leaf vein density and the area of xylem conduits in major veins, Nandou 12 exhibited significantly higher values than Guixia 7 under the ML and LL treatments. The major leaf vein density of Nandou 12 remained relatively stable across treatments, while that of Guixia 7 experienced a significant reduction of 11.4% and 15.0% under the ML and LL treatments compared to the HL treatment. Furthermore, a decrease in light intensity had a notable effect on increasing the distance between leaf veins and stomata. Specifically, under the ML and LL treatments, the distance from veins to stomata increased by 21.33% and 60.01% for Nandou 12 and by 31.50% and 53.59% for Guixia 7 in comparison to the HL treatment. The correlation analyses revealed significant positive correlations (P<0.05) between the hydraulic conductivity of soybean leaves and the density of minor leaf veins, the area of xylem conduits in major and minor veins. Conversely, a significant negative correlation (P<0.01) was observed between hydraulic conductivity and the distance from veins to stomata.【Conclusion】Light intensity exerts an influence on the leaf hydraulic conductivity by modulating the leaf vein structure of soybean. Under low light conditions, there is a reduction in leaf hydraulic conductivity in soybean; however, the coordination between leaf hydraulic conductivity and stomatal conductance is maintained to establish equilibrium between leaf water supply and demand as light intensity diminishes. The presence of a higher vein density under low light serves to abbreviate the distance required for water transport, thereby enhancing leaf water supply capacity. Consequently, this facilitates CO₂ diffusion and photosynthesis, representing an additional strategy employed by shade-tolerant soybean to acclimate to low-light environments.

Potato Tuber Skin Roughness Classification Analysis Based on Image Characteristics Recognition

TANG ZhenSan, YUAN JianLong, KANG LiangHe, CHENG LiXiang, LÜ Tai, YANG Chen, ZHANG Feng

Scientia Agricultura Sinica. 2023, 56(22): 4428-4440. doi:10.3864/j.issn.0578-1752.2023.22.006

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【Objective】The classification analysis of potato tuber skin roughness could provide the non-destructive testing methods for tuber appearance quality traits, which would establish the theoretical and practical base for the objective evaluation of tuber quality and high-throughput screening varieties.【Method】Seventy-nine potato varieties (lines) were selected as materials, and the images of tuber skin with and without bud-eyes were taken by camera. The tuber skin images were preprocessed using MATLAB R2016a software. Eight materials were randomly selected to compare the effect of image graying, enhancement and denoising using the correlation function indicators. The image characteristic parameters, angular second moment (ASM), entropy (ENT), contrast (CON) and correlation (COR) were extracted using the gray level co-occurrence matrix (GLCM), and the suitable distance (d) of GLCM were determined. The differences in two types of tuber skin image feature parameters were compared, and the set of tuber skin image features with less difference was selected for statistical analysis and classification recognition. The support vector machine (SVM) and backpropagation neural network (BPNN) models were constructed for tuber skin roughness classification, and the evaluation indexes of model grading accuracy were accuracy, precision, recall and harmonic mean, respectively. 【Result】The texture structure of tuber skin image after grayscale processing using the weighted average method was clear, and the evaluation value of image clarity was 2.5698±0.5959, which was significantly higher than that of the mean method (1.8035±0.4856) and the maximum method (1.0535±0.4088). The gray scale range of tuber skin image after histogram equalization enhancement was expanded from 100-200 to 0-200, which made the gray distribution wider. The salt noise denoising effect of tuber skin images using the median filter under 3×3 sliding windows was obvious, and the peak signal-to-noise ratio (PSNR) was maximum (28.6250±3.9784 Bp), which was significantly higher than that under 3×3 and 5×5 windows. Two types of tuber skin image feature parameters extracted by GLCM (d=4) were significantly different, and the set of tuber skin image features (without bud-eyes) with less difference was selected for statistical analysis and classification recognition. The results indicated that the variation coefficient of these parameters was varied significantly. The variation coefficient of contrast was the largest (0.40), followed by the angular second moment (0.24) and correlation (0.23), and the variation coefficient of entropy was the smallest (0.18). Using the feature set as the input variable of tuber skin classification model, the overall classification performance of SVM was higher than BP neural network, and the accuracy reached 87.5%. Especially, the prediction accuracy and recognizability of SVM for smooth and heavy hemp skins was the highest. The accuracy reached 100%, the recall reached 85.7% and 100%, and the harmonic mean reached 100% and 92.3%, respectively. 【Conclusion】The combination of the image processing techniques presented in this study and the GLCM extracted texture feature parameters could effectively characterize potato tuber skin roughness variations. The tuber skin roughness grading based on machine vision could be achieved by constructing SVM classification model, and the accuracy reached 87.5%.

Evaluation of Resistance and Analysis of Utilization Value of the Major Japonica Rice Varieties in Jilin Province Based on the Physiological Race Variation of Magnaporthe oryzae

LI li, SUN ling, ZHANG JinHua, ZOU XiaoWei, SUN Hui, REN JinPing, JIANG ZhaoYuan, LIU XiaoMei

Scientia Agricultura Sinica. 2023, 56(22): 4441-4452. doi:10.3864/j.issn.0578-1752.2023.22.007

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【Objective】Rice blast poses a serious threat to the safe production of rice in Jilin Province, China. Breeding and utilizing resistant varieties are the most economical, safe, and effective measures to control rice blast. On the basis of clarifying the physiological race forms, distribution, and pathogenicity of Magnaporthe oryzae, the resistance evaluation to rice blast and variety combination utilization value analysis for major japonica rice varieties in Jilin Province were carried out. It will provide a theoretical basis for rational variety deployment and the efficient utilization of resistant varieties.【Method】In the autumn of 2021, 62 dominant single spore strains were selected from 115 strains of M. oryzae collected and isolated from the main rice-growing areas of Jilin Province. These strains were then subjected to physiological race identification and pathogenicity analysis using seven Chinese differential varieties (CDVs). To assess the resistance of primary japonica rice varieties in Jilin Province, single pathogen inoculation at the seedling stage and field-induced resistance evaluation at various locations were carried out. Utilizing the results acquired from both the seedling stage and field identification, the resistance association coefficient (RAC) and virulence association coefficient (VAC) were used to analyze the disease resistance effect of various combinations of varieties.【Result】According to the resistance phenotype of different varieties (CDVs), 62 M. oryzae strains could be divided into 7 groups and 22 physiological races. The dominant physiological groups were ZG and ZA, with frequencies of 35.48% and 32.26%, respectively. The dominant physiological races were ZG1, ZA1, and ZA17, with frequencies of 35.48%, 9.68%, and 8.06%, respectively. The moderately virulent races with a pathogenicity rate of over 40% were ZA49, ZA1, ZB15, ZB23, and ZC15. The results of single-strain inoculation at the seedling stage of the main japonica rice varieties in Jilin Province showed that 48.89% of the tested rice varieties had a seedling blast resistance frequency above 80%. According to the field evaluation of panicle blast resistance, 14 varieties demonstrated moderately resistant (MR), accounting for 15.56% of the evaluated varieties. Additionally, 27 varieties exhibited moderately susceptible (MS), representing 30% of the evaluated varieties. Furthermore, 35 varieties were classified as susceptible (S), making up 38.88% of the evaluated varieties. Finally, 14 varieties exhibited highly susceptible (HS), representing 15.56% of the total. The analysis of disease resistance through the combinations of varieties revealed that the combinations simultaneous presence of a high RAC and low VAC have a good application prospect.【Conclusion】The physiological race structure of the 62 M. oryzae strains in Jilin Province is complex and diverse, with different dominant physiological races in different rice-growing regions. Additionally, the major japonica rice varieties in Jilin Province exhibit strong resistance and a broad resistance spectrum against the rice blast. Moreover, some combinations of varieties possess significant application value.

Screening, Identification and Control Efficacy Analysis of Trichoderma Strains Against Maize Pythium Stalk Rot

GUO Ning, SUN Hua, MA HongXia, LIU ShuSen, ZHANG HaiJian, SHI Jie, ZHENG XiaoJuan, DONG YueGuang

Scientia Agricultura Sinica. 2023, 56(22): 4453-4466. doi:10.3864/j.issn.0578-1752.2023.22.008

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【Objective】The objective of this study is to screen Trichoderma strains which have inhibitory effect on the Pythium spp. causing maize stalk rot, and to clarify their taxonomic status, control efficacy and antifungal mechanism. This study will provide important resources for the research and development of biocontrol agent against Pythium stalk rot.【Method】For the antagonistic strains screening, the inhibitory effect of Tichoderma strains on P. inflatum, P. arrhenomanes and P. aristosporum was tested by measuring the mycelia growth. The taxonomic status of Tr21 was determined by morphological and molecular characteristics. The effect of Tr21 on the mycelia morphology of Pythium spp. was observed in the laboratory. In order to analyze the effect of Tr21 fermentation broth on the membrane permeability of Pythium spp., propyridine bromide (PI) dye solution was used to stain, and the absorbance values of protein and nucleic acid in mycelia supernatant at different treatment times were detected. The effect of Tr21 fermentation broth on germination characteristics of maize seeds was tested by seed soaking with different concentrations of fermentation broth. The control efficacy of Tr21 on stalk rot was confirmed through greenhouse pot and field inoculation experiments.【Result】From the 109 strains of Trichoderma spp., seven strains were screened with antagonistic activity against P. inflatum, P. arrhenomanes and P. aristosporum, and the inhibition rate was above 60%. The inhibition rate of Tr21 to three Pythium species reached 100%, the inhibition rate of 5×, 10× and 20× diluent to three Pythium species reached 100%, and the inhibition rate of 50× diluent to three Pythium species was also more than 55.56%. Tr21 strain was identified by morphological and molecular biology as T. afroharzianum. The results of microscopic observation showed that the fermentation broth of Tr21 could cause mycelial malformations, such as rough mycelia, increased mycelial branching, shortened nodes, and overflow of mycelia contents. The result of PI fluorescence stain showed that the cell membrane of three Pythium species was damaged by Tr21 fermentation broth, and the PI dye was more likely to penetrate the damaged cell membrane into the mycelium and stain the mycelia red. The results of nucleic acid and protein leakage showed that the absorbance values of the mycelia treated by the fermentation broth changed greatly. After treatment for 5 h, the OD₂₆₀ increased by 0.08 and OD₂₈₀ increased by 0.10, 0.11 and 0.10, respectively, indicating that the membrane of the mycelia was damaged, leading to the overflow of mycelia contents. The different concentrations of Tr21 fermentation broth had no effect on the germination characteristics of maize seeds, and the 20× diluent had the best effect on germination and growth of seeds. The results of pot experiment showed that 5× diluted fermentation broth of Tr21 had the best control efficacy on Pythium stalk rot caused by three Pythium species, which was 60.67%, 63.15% and 59.66%, respectively. The control efficacy on Pythium stalk rot of 5× diluent was the highest, reaching 82.25%, with a mass ratio of 1﹕100 (5× diluent to seed).【Conclusion】An effective T. afroharzianum strain Tr21 was obtained for preventing and controlling of maize Pythium stalk rot. The fermentation broth of Tr21 can lead to mycelia malformation, breakage, cell membrane damage and contents leakage, etc. In conclusion, the T. afroharzianum strain Tr21 is a promising biocontrol microbial.

Standardized Establishment and Improvement of Accounting System of Agriculture Greenhouse Gas Emission

ZHANG WeiJian, SHANG ZiYin, ZHANG Jun, YAN ShengJi, DENG AiXing, ZHANG Xin, ZHENG ChengYan, SONG ZhenWei

Scientia Agricultura Sinica. 2023, 56(22): 4467-4477. doi:10.3864/j.issn.0578-1752.2023.22.009

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Agriculture is not only the dominant source of human food and clothing, but also the potential sector of global anthropogenic greenhouse gas (GHG) emissions and mitigation, especially methane (CH₄) and nitrous oxide (N₂O). To standardize GHG accounting is an urgent need for agricultural carbon emission inventory compilation, carbon trading of emission reduction verification, carbon reduction subsidy and low-carbon agricultural product certification, as well as the basis for the policy making and technology selection of agricultural carbon reduction and sequestration, which is of great significance for the green-low-carbon and high-quality development of agriculture. Based on a systematic review of the relevant global specifications, guidelines, methodologies and standards of agricultural GHG accounting, this paper aimed to address the problems of imperfect monitoring and reporting systems, unsystematic accounting and calculating methods, and uncertain accounting results, by providing the following four suggestions for the establishment and improvement of agricultural GHG accounting systems. Firstly, we should further establish and improve the institutional system of the accounting system, to better clarify the subject of the main responsibility. On the existing basis of China's agricultural statistics and non-point source pollution monitoring and reporting systems, we should strengthen the construction of agricultural GHG emission monitoring (M), reporting (R) and verifying (V) system (i.e. MRV system), and supplement and improve the policy making and institutional setting, so as to clarify the main responsibilities of agricultural GHG statistical accounting and carbon reduction and sequestration. Secondly, we should further supplement and improve the accounting standards and methodologies. According to the newly issued international standards and methodologies, and the actual situation of China’s agricultural production and future development, we need to revise the agricultural components of China's Guidelines of Provincial GHG Emission Inventories. For example, the farmland carbon sequestrations of biochar application, ecological farm and well-facilitated farmland construction, photovoltaic farms and crop straw comprehensive utilization, as well as the carbon emissions of lime and urea application, ruminant livestock feeding and freshwater aquaculture, need to be supplemented into the guidelines. The accounting standards and methods of agricultural indirect GHG emissions need to be revised, and some new CCER (Chinese certified emission reduction) methodologies need to be developed for agricultural carbon trading. Thirdly, the database needs to be further renewed and upgraded. We need to strengthen scientific and technological innovations and accounting data accumulation of agricultural carbon reduction and sequestration, to renew and upgrade the basic data, action data and emission factor data of the existing accounting systems in combination with field monitoring, model estimation and literature synthesis. Fourthly, at last, it is also necessary to develop the application software supporting the accounting standards and methodologies, and carry out science popularization, technical training and application demonstration. Our suggestions could provide the references for the guideline revision of agricultural greenhouse gas emission inventories, and supports to the methodology development for trading verification of agricultural voluntary emission reduction and carbon footprint assessment of low-carbon agricultural product certification.

Optimization Path of the Ecological Compensation Mechanism for Conservation Tillage in the Northeast Black Soil Region

ZHOU Ying, YANG Peng, WANG LiGang, LEI QiuLiang, ZHANG YaNan

Scientia Agricultura Sinica. 2023, 56(22): 4478-4489. doi:10.3864/j.issn.0578-1752.2023.22.010

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In the new period, the protection of cultivated land quality in the black soil region should be the primary task to strengthen the “ballast stone” of national grain security, and to consolidate and to improve the comprehensive grain production capacity in Northeast China. Conservation tillage, while being a key technology for protecting and improving the quality of black soil, has practical constraints on its large-scale application at the policy and subject levels. The aim of this study was to understand scientifically the shortcomings of ecological compensation policies for conservation tillage in the black soil areas of Northeast China and to explore the optimization paths for the compensation policies for conservation tillage technology with a problem-oriented approach, which could provide a scientific basis for making up for the shortcomings of the system and achieving precise policy implementation. The subsidy standard of corn straw returning technology in northeast black soil region was summarized, the practical constraints of subsidy policy were qualitatively analyzed, and the main obstacle factors of current conservation tillage support policy were clarified by comprehensively utilizing literature analysis, normative analysis, and inductive deduction methods combined with field research. Three core issues were included the unclear direction of compensation policy, the unscientific basis for standard compensation pricing, and the incomplete management mechanism and institutional system. In response to the practical difficulties that ecological compensation for conservation tillage faces in black soil areas, three paths for optimizing the policy mechanism were proposed: first, the empowerment and systematization were strengthened to activate the subjectivity path of conservation tillage, and the supporting policies were established from two levels: improving empowerment positioning and cultivating new business entities. Second, a collaborative, quantitative, and standardized compensation evaluation method was constructed for environmental-economic attributes, external effects and subjective effects generated by technology were used as the theoretical basis for pricing compensation standards, and then an evaluation method system was established with integrated multiple methods, including alternative market technology, hypothetical market technology, and econometric statistical analysis. Third, a high-quality digital management and differentiated ecological compensation system was prepared for building an open and shared big data management platform, creating an ecological market picture of multi-agent collaboration and mutual benefit, and establishing multi-agent conservation tillage collaboration and a differentiated ecological compensation mechanism. In order to adapt to the new requirements for technical innovation and organizational changes in conservation tillage, a scientific evaluation system was established for the application of green technologies in conservation tillage agriculture, and optimizing the vertical management and vertical compensation mechanism, guiding enterprises and social capital to participate in market regulation, and building an ecological management system barrier for the protection and improvement of cultivated land quality in black soil areas, which provided references for optimizing the ecological compensation policy.

Development and Transferability of EST-SSR Markers Based on Transcriptome Data from Asparagus officinalis

YI ZeHui, ZHAO Jing, MAO LiPing

Scientia Agricultura Sinica. 2023, 56(22): 4490-4505. doi:10.3864/j.issn.0578-1752.2023.22.011

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【Objective】The aim of this study was to clarify the distribution law of SSR loci across the transcriptome of Asparagus officinalis, to develop highly informative EST-SSR markers and to analyze their transferability, so as to provide the tools for phylogenetic analysis, functional gene mining and molecular marker-assisted breeding of asparagus plants.【Method】Based on the RNA-seq data of 15 asparagus roots obtained from the previous stage by our research group, MISA software was used to retrieve SSR loci, and Primer 3.0 software was employed to design primers in batches. Then, the ineffective primers were eliminated by performing batch e-PCR with TB-tools software and one-to-one e-PCR with the Primer-blast programme. The information of EST-SSR markers (such as gene id, physical location, and potential function) was obtained by comparison with the genome of asparagus officinalis. The DNA of 9 A. officinalis varieties, 7 A. setaceus varieties, 5 A. cochinchinensis varieties, and 3 A. umbellatus varieties were used as templates, and 50 pairs of randomly synthesized primers were used as primers to detect the effectiveness, polymorphism and transferability of the primers developed.【Result】A total of 36 590 simple sequence repeats (SSRs) loci distributed in 30 229 unigenes with a frequency of 4.78% and an average distance of 9.17 kb were identified based on data from 15 root transcriptomes of A. officinalis. The SSRs were unevenly distributed in the 10 chromosomes, with the highest number in chromosome 7 (4 642) and the highest density in chromosome 3 (37.86 SSRs/Mb). The SRRs were distributed from di- to hexa-, with tri- (46.92%) and AG/CT (16.58%) as the most abundant repeat type and predominant repeat motif, respectively. A total of 19 695 pairs of EST-SSR primers were successfully designed, and 15 147 pairs ineffective primers were eliminated by e-PCR. Among them, 3 085 pairs ineffective primers didn’t produce any amplification products, 10 102 pairs produced severely inconsistent amplification products in terms of fragment size, 1 289 pairs had unknown physical positions in the genome, 402 pairs gave other amplification products of similar size to the target fragments, and 269 pairs generated amplification products without SSRs. Based on 2 517 EST-SSR markers located in the gene region developed in this study, the chromosome density distribution map was constructed, with a total coverage length of 1 125.51 Mb and an average distance of 447.16 kb. The potential functions of these markers were involved in many aspects, such as yield, quality, stress resistance, and so on. All 50 pairs of randomly synthesized primers could amplify target bands clearly, of which 36 pairs were polymorphic, and the average polymorphic information content was 0.330. These markers could be used in three other species of Asparagus: the transferability to A. cochinchinensis, A. setaceus, and A. umbellatus were 100%, 92%, and 88%, respectively. Cluster analysis based on the EST-SSR alleles grouped the 24 accessions into four clusters that corresponded to the species of A. officinalis, A. setaceus, A. cochinchinensis, and A. umbellatus.【Conclusion】In this study, 2 517 highly informative EST-SSR markers of asparagus were successfully developed, and the effective amplification rate was 100%. The total coverage length of the physical map was 1 125.51 Mb, and the average distance was 447.16 kb, which could be used for phylogenetic analysis of asparagus and related species. Moreover, it provided a reference for the development of EST-SSR markers in other species.

Screening Regulatory Genes Related to Luffa Fruit Length and Diameter Development Based on Transcriptome and WGCNA

CHEN MinDong, WANG Bin, LIU JianTing, LI YongPing, BAI ChangHui, YE XinRu, QIU BoYin, WEN QingFang, ZHU HaiSheng

Scientia Agricultura Sinica. 2023, 56(22): 4506-4522. doi:10.3864/j.issn.0578-1752.2023.22.012

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【Objective】 The aim of this study was to identify the co expression modules of luffa fruit length and diameter development and to screen key regulatory genes, so as to provide the theoretical basis for subsequent research on the molecular mechanism of fruit shape control in luffa. 【Method】 The luffa fruits in 9 fruit development stages (2 days before anthesis, and 0, 2, 4, 6, 8, 10, 15, and 20 days after anthesis) were applied as research materials. The fruit length and diameter of each stage were measured. The WGCNA method was used to jointly analyze transcriptome and fruit length and diameter data, to identify co-expressed gene modules of fruit length and diameter development, and to screen out key regulatory genes.【Result】A total of 14 co expression modules were identified by WGCNA, among which two modules (Turquoise and Lightpink4) were significantly correlated with fruit length and diameter (absolute value of correlation coefficient=0.9); Turquoise module was significantly positively correlated, while Lightpink4 module was significantly negatively correlated. KEGG enrichment analysis found that the Turquoise module was significantly enriched in endocytosis and phenylpropanoid biosynthesis pathways, which were closely related to fruit enlargement and growth regulation, and could be used as a key gene module for studying fruit length and diameter in luffa. According to the connectivity and functional annotation of genes in Turquoise module, ten key regulatory genes were screened, including xyloglucan endotransglucosylase/hydrolase gene XTH23, actin-depolymerizing factor gene ADF2, chaperone protein gene DnaJ10, expansin gene (EXPA1, EXPA4 and EXLA5), kinesin gene kinesin-13A, auxin response genes SAUR21, and Aux/IAA11. The RT-qPCR results showed that the expression levels of ten regulatory genes significantly increased after the fruit entered the rapid growth period (8 day after anthesis), with an increase of 2-50 times approximately. Through constructing a gene interaction network, it was found that some candidate genes interacted with the WRKY, bHLH, and HSF transcription factor families.【Conclusion】The Turquoise module, an important co expression module of luffa fruit length and diameter was obtained, and ten potential candidate genes for luffa fruit shape control were screened. It was found that luffa fruit length and diameter development regulation mainly involved the processes of cell wall reconstruction, cell development and differentiation, and auxin regulation.

Mechanism of Hydration Environment/Magnetic Field Effects on the Oxidative Stability of Myoglobin

DENG YuShi, XIA MinQuan, MA Jing, ZHOU YuanHua, SUN WeiQing

Scientia Agricultura Sinica. 2023, 56(22): 4523-4531. doi:10.3864/j.issn.0578-1752.2023.22.013

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【Objective】To investigate the mechanism of action of hydration environment affecting the oxidative stability of myoglobin (Mb) in the presence of a magnetic field, and to provide a basis for improving the oxidative stability of Mb. 【Method】Two magnetic field environments of low intensity (3 mT) and high intensity (12 mT) were set up at 4 ℃, the magnetic field treated Mb aqueous solution, Mb powder, and deionized water (the magnetic field treated deionized water and then dissolved Mb), respectively, and the magnetic field treatment time was 1 h. The Mb aqueous solution without magnetic field treatment was used as the control. The oxidative stability properties of Mb were analyzed by the relative content of high iron myoglobin, heme iron content and the variation of UV absorption bands, while the changes of Mb secondary structure, tertiary structure and porphyrin iron structure were analyzed by circular dichroism, and Raman spectroscopy and fluorescence spectroscopy techniques were employed to investigate the mechanism of the effect of magnetic field on the oxidative stability of Mb. 【Result】Both magnetic field treatment of Mb powder directly and magnetic field treatment of solvent water followed by dissolution of Mb had no significant effect (P>0.05) on the relative content of methemoglobin, while both 3 mT and 12 mT magnetic field treatment of Mb aqueous solution significantly increased the relative content of methemoglobin. The results of heme iron content and heme Shore band UV absorption showed that the heme porphyrin ring structure was sensitive to the magnetic field environment, and the magnetic field of different intensities had significant damage to the Mb heme structure, while the high intensity magnetic field environment had relatively greater damage to the porphyrin ring structure. Mb tertiary and secondary structure results showed that both 3 mT magnetic field treatment of solvent water before dissolving Mb, and 3 mT and 12 mT magnetic field treatment of Mb aqueous solution significantly promoted the unfolding of Mb secondary structure and oxidative damage of tryptophan and tyrosine residues of side chain groups. Raman spectroscopy results showed that 12 mT magnetic field treatment of Mb aqueous solution induced the cross-linking of Mb through disulfide bonds. 【Conclusion】Hydration in Mb aqueous solution directly affected the effect of magnetic field on the oxidative properties of myoglobin, and magnetic field treatment promoted the oxidation of Mb central iron as well as heme porphyrin ring, probably because the magnetic field changes the physical properties of water molecules, such as dielectricity and degree of ionization, as well as the hydrogen bonding state between Mb and water, which further affected the structure of Mb with the unfolding of α-helix structure and the exposure of side chain groups, accelerating the destruction of heme structure and loss of heme iron, and promoting the oxidation of central iron.

Effects of Dietary Fiber Level on Intestinal Barrier Function, Colonic Microbiota and Metabolites in Pigs

LIU Chang, CUI ZiXu, ZUO Zhou, YUN HongMei, NIU Jin, YANG Yang, GUO XiaoHong, LI BuGao, GAO PengFei, ZHAO Yan, CAO GuoQing

Scientia Agricultura Sinica. 2023, 56(22): 4532-4551. doi:10.3864/j.issn.0578-1752.2023.22.014

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【Objective】 Adding fiber raw materials is one of the effective methods to reduce the feed costs. The aim of this study was to investigate the effects of different fiber level diets on the intestinal health of Mashen (MS) pig and Duroc × Landrace × Large (DLY) pig, so as to provide the basis for the rational use of fiber. 【Method】 In this study, 80 MS pigs and 80 DLY pigs with an initial body weight of (20 ± 0.5) kg were used as test objects (half of the sows and half of the boars). MS and DLY pigs were divided into four groups each assigned to different diets, with five replicates per treatment and four pigs per replicate. When 0%, 9.35%, 18.64% and 28.03% soybean hulls were added to the corn soybean meal basal diet, the NDF content was 9% (9N), 13.5% (13.5N), 18% (18N) and 22.5% (22.5N), respectively. The test lasted for 30 days. 【Result】 For MS pigs, the content of IL-10 in ileum of 18N group and TNF-α in cecum of 13.5N and 22.5N groups was significantly decreased (P<0.05). The number of goblet cells in jejunum, ileum and caecum was increased with the increase of fiber level. In colon, the number of goblet cells and the expression level of MUC2 were significantly increased in 13.5N and 18N groups (P<0.05). The expressions of Claudin-2, Occludin, E-cadherin, ZO-1, and ZO-2 of 18N and 22.5N groups were increased significantly (P<0.05). The abundance of Escherichia-Shigella in 13.5N, 18N and 22.5N groups were decreased significantly (P<0.05), while the abundance of Lactobacillus in 13.5N group, Prevoteaceae_NK3B31_group in 18N group and Methanobrevibacter in 22.5N group were increased significantly (P<0.05). Palmitic acid, stearic acid, lauric acid and capric acid in 13.5N, 18N and 22.5N groups were significantly increased (P<0.05), while lithocholic acid and cholic acid were significantly decreased (P<0.05). Differential metabolites were mainly concentrated in the pathways related to lipid metabolism and carbohydrate digestion and absorption. For DLY pigs, the TNF-α content in cecum of 13.5N group was significantly increased (P<0.05). The number of goblet cells in each intestinal segment of 13.5N, 18N and 22.5N groups was significantly higher than those of 9N group (P<0.05). The expression level of colonic MUC2 was increased significantly in 13.5N and 18N groups (P<0.05) and decreased significantly in 22.5N group (P<0.05). The expression levels of Claudin-2, Occludin, E-cadherin and ZO-1 in 18N and 22.5N groups were decreased significantly (P<0.05). The abundance of Methanobrevibacter in 13.5N group, Streptococcus in 18N group and Lachnospira in 22.5N group was significantly increased (P<0.05). L-tyrosine, L-glutamic acid, L-pyroglutamic acid, phenylalanine and other derivatives in 13.5N, 18N group were significantly increased (P<0.05), while significantly decreased (P<0.05) in 22.5N group. Differential metabolites were mainly concentrated in amino acid metabolism pathway. In MS pigs and DLY pigs, there was a strong correlation between microflora and intestinal barrier related genes, as well as tryptophan and bile acid metabolites. 【Conclusion】 Improving the dietary fiber level could strengthen the intestinal barrier function of pigs, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, regulate the metabolism of lipid and amino acids in the colon, affect the metabolism of short-chain fatty acids, tryptophan and bile acids through the microbial pathway, and promote intestinal health.

Modeling Net Energy Requirement of 1-60 Days Old Wenchang Hen

XU XiaoJing, WEI LiMin, ZHAO ShaoMeng, ZHAO GuiPing, ZHANG MinHong, FENG JingHai

Scientia Agricultura Sinica. 2023, 56(22): 4552-4560. doi:10.3864/j.issn.0578-1752.2023.22.015

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【Objective】The aim of this study was to determine the net energy requirements of Wenchang hens aged 1-60 days, so as to provide a scientific basis for efficient breeding of Wenchang hens. 【Method】The net energy requirements of growth (NEg) and maintenance (NEm) of Wenchang hens aged 1-30 and 31-60 days were estimated by factorial method. In experiment 1, three hundred 1-day-old healthy Wenchang chicken chicks were randomly divided into 6 replicates and routinely fed until 56 days of age. The body weight (BW) and net body energy (BE) of Wenchang hens were measured every week, and then a linear regression model between BW and BE was established. The slope was the NEg of Wenchang hen. The NEm of Wenchang hens was investigated in two phases. Two hundred and forty healthy Wenchang hens with similar BW were selected at 1 and 31 days of age, respectively. The birds were randomly divided into four treatment groups with six replicates. The four treatment groups were fed the same metabolic diets, which were ad libitum, 20%, 40% and 60% restricted. Test period was 30 days per period. During the middle stage of the experiment (14 and 44 days old), excreta were collected continuously for 3 days to determine dietary apparent metabolizable energy (AME) at different feeding levels; at the beginning and end of each stage, retained energy (RE), metabolizable energy intake (MEi) and heat production (HP, HP = MEi-RE) of Wenchang hens were measured, and a regression model between the logarithm of HP and MEi was established: lg(HP) = a + b×MEi; the HP when MEi = 0 was calculated, which was the NEm of Wenchang hens.【Result】With the increase of age, BW, daily gain (ADG), feed intake (ADFI), feed conversion ratio (G/F) and BE of Wenchang hens significantly increased (P<0.001). The NEg value was 8.20 and 12.07 kJ·g^-1 in Wenchang hens aged 1-30 days and 31-60 days, respectively. Restriction significantly increased the AME value of diet (P<0.001); MEi and HP significantly decreased with the decrease of food intake (P<0.001); there was a significant logarithmic regression relationship between MEi and log HP (R²= 0.91 and 0.90, P<0.001), and the NEm value at 1-30 days and 31-60 days of age was 300.61 and 398.11 kJ·kg^-1BW^0.75·d^-1, respectively. 【Conclusion】The NEg and NEm of Wenchang hens were different at different growth stages, and the daily net energy requirement of Wenchang hens could be predicted according to BW and ADG. The prediction models were: NE_1-30d= 300.61×BW^0.75+ 8.20×ADG and NE_31-60d = 398.11×BW^0.75 + 12.07×ADG.

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