Scientia Agricultura Sinica

QTN Mining and Candidate Gene Screening of Upland Cotton (Gossypium hirsutum L.) Seed-Related Traits

BAI BingNan, QIAO Dan, GE Qun, LUAN YuJuan, LIU XiaoFang, LU QuanWei, NIU Hao, GONG JuWu, GONG WanKui, ELAMEER ELSAMMAN, YAN HaoLiang, LI JunWen, LIU AiYing, SHI YuZhen, WANG HaiZe, YUAN YouLu

Scientia Agricultura Sinica. 2024, 57(15): 2901-2913. doi:10.3864/j.issn.0578-1752.2024.15.001

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【Objective】Exploring the genetic loci and related genes that control cottonseed size traits to lay a foundation for subsequent study on the molecular mechanism cottonseed size formation. 【Method】The upland cotton recombinant inbred line (RIL) population composed of 300 lines was used as the research material. Seven phenotypic traits including cottonseed index (SI), seed length-cutting acreage (SLA), seed length-cutting perimeter (SLP), seed length (SL), seed width (SW), length-width ratio (LWR) and seed roundness (SR) were evaluated in four environments. The RIL population was genotyped by liquid phase chip strategy. The high-quality single nucleotide polymorphism (SNP) markers and phenotypic data were subjected to perform genome-wide association study (GWAS), and quantitative trait nucleotides (QTNs) associated with cottonseed size-related traits were mined. The genetic effects of QTNs were analyzed to identify candidate genes. 【Result】Seven cottonseed size-related traits showed a continuous normal distribution in four environments, which expressed a sizable phenotypic variation. The coefficient of variation ranged from 1.82% to 10.70%. The influencing effect on trait formation were basically as genotype>environment>genotype × environment, indicating suitability for GWAS analysis of these results. Correlation analysis showed that the seed index was significantly correlated with SLA, SLP, SL and SW, and LWR was significantly correlated with SR, indicating the possible existence of pleiotropic loci. GWAS was performed using the 3VmrMLM model, and a total of 47 QTNs were associated with these seven traits. A total of 11 QTNs were associated on chromosome A07, of which three physical loci in the region of 71.99-72.87 Mb, A07:71993462, A07:72067994 and A07:72198802 were very close and simultaneously associated with SI, SLA, SLP, SL and SW in four environments. The average value of R2 between markers was>0.8 (P<0.001), showing a large linkage disequilibrium. Genetic effect analysis showed that there were two haplotypes in this region. Among these cottonseed size relating traits, haplotype Ⅱ and haplotype I were significantly different, indicating that these loci directly affected cottonseed size traits and could be used for molecular marker-assisted selection. The expression patterns of the genes in the interval were analyzed using TM-1 transcriptome data. The results revealed that Gh_A07G1767 was preferentially expressed and Gh_A07G1766 specifically expressed at the stage of cottonseed development. These results speculated that these genes may play an important role in the growth and development of cottonseed.【Conclusion】47 QTNs were identified, and two candidate genes related to cottonseed development were screened.

Identification of Candidate Genes Controlling SSCLD by Utilizing High-Generation Segregating Populations RNA-seq

CHEN WenJie, CHEN Yuan, WEI QingYuan, TANG FuYue, GUO XiaoHong, CHEN ShuFang, QIN XiaYan, WEI RongChang, LIANG Jiang

Scientia Agricultura Sinica. 2024, 57(15): 2914-2930. doi:10.3864/j.issn.0578-1752.2024.15.002

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【Objective】 Severe SSCLD can lead to about 40% yield reduction of soybean. In this study, candidate genes associated with controlling SSCLD were identified by HGRNA-seq technology to provide data support for revealing the molecular mechanisms of SSCLD.【Method】Two parents with SSCLD resistance difference and their derived F_2:7 lines were used as the experimental materials. Two parents and 12 F_2:7 lines (7 crinkled leaves and 5 normal leaves) were separately subjected to re-sequenced and transcriptome sequencing. Mixed pool method was carried out for gene localization by using SNP/InDel data from the parents and their progenies. GO and KEGG functional annotation and enrichment analysis was carried out by using differentially expressed genes (DEGs) from RNA-seq. 7 KASP molecular markers were developed in the vicinity of localization interval, and 230 F₂ populations were used to construct a localized linkage map to verify the localization results of the transcriptome data. Candidate genes controlling SSCLD were screened by combining gene mapping and transcriptome analysis.【Result】The gene controlling SSCLD named CL12 was located on chromosome 12 within a 1 473 464 bp interval by using mixed pool method, ranging from 39 231 651 bp to 40 705 115 bp. At the same time, the gene was localized in the 1 205 020 bp interval from 39 743 275 to 40 948 295 bp by using the F₂ population, which was basically consistent with the results of the mixed-pool method. The GO annotation results showed that the metabolic processes included immune system processes and responses to stimuli, and cellular components were mainly related to membranes, etc. The KEGG annotation results showed that the biosystem pathways mainly included plant-pathogen interaction and environmental adaptation, etc. GO-enriched DEGs were mainly related to the activity of transmembrane receptor proteins, protein phosphorylation, and signaling receptors, etc., KEGG enriched DEGs were mainly related to plant-pathogen interaction and MAPK signaling pathway. Combined with the characteristics of the causal factors of SSCLD, genes within the candidate interval associated with disease resistance which had non-synonymous mutations in the coding exons, or showed differential expression were selected as candidate genes for SSCLD resistance. GLYMA_12G223100, GLYMA_12G223900, GLYMA_12G224100, GLYMA_12G231800 and GLYMA_12G233000 were finally identified as the candidate genes controlling SSCLD by qRT-PCR.【Conclusion】HGRNA-seq realized the combination of RNA-seq and BSA-seq, and successfully mined the candidate genes controlling SSCLD.

Combining Ability Analysis on Quality Traits and Breeding Potential Evaluation of 23 Waxy Maize Lines from Laos

ZHU ChunTao, REN DanDan, LIU ZhengCen, LIU ChangChuang, LIU RuiQi, ZHENG HongJian, HU ErLiang, LIN HaiJian, LI JingWei, LU YanLi, WANG QingJun

Scientia Agricultura Sinica. 2024, 57(15): 2931-2945. doi:10.3864/j.issn.0578-1752.2024.15.003

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【Objective】High-quality is the primary target for waxy maize breeding. The aim of this study is to screen lines with high general combining ability and excellent hybrids with high-quality traits, which will clarify the breeding potential of these lines from Laos and lay a foundation in breeding high-quality waxy maize varieties.【Method】Using the Maize6H-60K chip, high quality genotype data of 33 waxy maize materials were obtained for cluster analysis. Combined with the iodine staining experiment and molecular detection of waxy genes, these types of waxy genes from early-generation maize lines from Laos were identified. Through the incomplete diallel cross experiment, phenotype identification and combining ability analysis of quality and other traits of 439 combinations were carried out to clarify the breeding potential of waxy maize lines with early generation from Laos. An experiment of quality traits evaluation with multi-person was conducted on these selected combinations with advantages to identify the most promising high-quality waxy maize hybrids. 【Result】Clustering analysis was conducted based on 56 626 high quality SNP, and the results showed that these lines from Laos and domestic materials belong to different branches and can be clearly distinguished. All 23 lines with early generation from Laos were waxy maize, among which 16 lines belonged to the wx-D10 type, three lines belonged to the wx-D7 type, and the other four lines were unknown types. The heritability of quality traits of waxy maize materials from Laos is low, ranging from 0.14 to 0.35, which is suitable for selection in higher generations. The average stewing quality of 439 combinations was significantly higher than that of the control Jingkenuo2000 and Shinuo2, but there was no significant difference from Yunuo7. The general combining ability effects of all quality traits of waxy maize lines F02, F22, F25, and F28 were positive, which could be used to improve quality traits of domestic waxy maize in China. Furthermore, these combinations M02×F02 and M22×F22 were evaluated as excellent in multi-person quality traits evaluation experiments by approximately 86% of all tasters.【Conclusion】These local varieties of waxy maize from Laos have rich genetic variation, with a certain genetic distance from domestic waxy maize inbred lines. There may also exist new alleles of waxy gene in these lines from Laos. In addition, these waxy maize lines from Laos have obvious advantages in quality traits, including waxy quality, pericarp thickness and other traits, which can be used as valuable germplasm for improving the quality traits of domestic waxy maize in China.

Effects of Foliar Application Uniconazole on Culm Morphological Characters, Anatomical Traits and Stem Lodging Resistance of Hybrid Indica Rice Under Low Light Stress

ZHANG WuJun, DUAN XiuJian, LI MaoYu, LUO Xia, LIU QiangMing, TANG YongQun, LI JingYong, YAO Xiong

Scientia Agricultura Sinica. 2024, 57(15): 2946-2963. doi:10.3864/j.issn.0578-1752.2024.15.004

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【Objective】The objective of current study was to investigate the regulation mechanism of endogenous hormones, culm anatomical and morphological traits and its relationship with lodging resistance of hybrid indica rice as affected by foliar application uniconazole under shading condition. The study results could provide theoretical basis and technical support for stable and abundant hybrid indica rice yield and emergency chemical regulation cultivation under poor light stress in Sichuan Basin. 【Method】A split-split plot field experiment was carried out in Yongchuan, Chongqing from 2018 to 2019. Shading was selected as the main plots, namely, normal light treatment (NL) and shading treatment with decreased incident light by 50% (S). The foliar application uniconazole levels were used as splits-plots, and three uniconazole levels were set: 0 (U₀), 40 mg·L^-1 (U₄₀), and 80 mg·L^-1 (U₈₀), and the varieties were used as split-split plots, namely Yuxiang203 (YX203) and Cliangyouhuazhan (CLYHZ), respectively. The study focused on analyzing the effects of foliar application uniconazole on stem lodging resistance of hybrid indica rice plant under shading condition, and further investigating the changes of culm morphological, anatomical traits and endogenous hormones and its relationship with stem mechanical strength. 【Result】Shading and uniconazole treatments had significant effects on culm morphological and anatomical traits, changes of endogenous hormones and stem lodging index (LI) and its physical parameters in two hybrid indica rice varieties. Compared with NL, S treatments significantly raised LI owing to significant reduction of breaking resistance (M), even though bending moment by whole plant (WP) also decreased, especially for YX203. Further, S treatments significantly lowered thickness of mechanical tissue, area of small and large bundle vascular in basal culm internode. And, cell width was decreased slightly, but ratio of cell length to cell width was increased to a certain extent, thus reduced culm wall thickness and stem mechanical strength consequently. Under shading stress, foliar application uniconazole significantly decreased stem LI of two hybrid indica rice varieties, which attributed to lower WP and higher M values, especially for YX203. Foliar application uniconazole reduced plant height and gravity center height by shorter length of lower internodes. And further, endogenous hormones including GA₁₊₃and IAA content during culm formation stage was declined, induced reduction of cell length and ratio of cell length to cell width and thereby, contributed to shorter lower internodes. Besides, endogenous hormones Z+ZR and IP+IPA content were increased during culm early development, the cells size showed smaller and denser, cell layer of mechanical tissue and cell number of bundle vascular sheath were also increased, and thus improved small and larger bundle vascular number and area and stem mechanical strength. The correlation analysis showed that cell length, ratio of cell length to cell width, thickness of mechanical tissue and area of small and large bundle vascular were significantly and negatively correlated to LI, but positively correlated to M. The trend were consistent in both YX203 and CLYHZ.【Conclusion】Foliar application uniconazole declined cell length, ratio of cell length to cell width and length of basal internodes primarily owing to reduction of GA₁₊₃and IAA content, and increased cell layer of mechanical tissue, cell number of bundle vascular sheath and cell density in parenchyma tissue by increasing Z+ZR and IP+IPA content, and raised thickness of mechanical tissue, area of small and large bundle vascular and thereby, improved stem mechanical strength and lodging resistance of hybrid indica rice under poor light stress.

Effects of Sowing Date Adjustment on Yield and Quality of Winter Wheat and Summer Maize in Northern Area of North China

ZHAO HuaRong, ZHOU GuangSheng, QI Yue, GENG JinJian, TIAN XiaoLi

Scientia Agricultura Sinica. 2024, 57(15): 2964-2985. doi:10.3864/j.issn.0578-1752.2024.15.005

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【Objective】Based on the field staging experiments, this study revealed the different responses of winter wheat and summer maize sowing date adjustments in growth and development, photosynthetic physiological characteristics, grain filling, yield formation and quality to climate warming in northern area of North China, providing scientific basis for agricultural production measures to cope with climate change in North China Plain.【Method】Different sowing date experiments of winter wheat and summer maize have been conducted at Hebei Gucheng Agricultural Meteorology National Observation and Research Station in northern area of North China from 2017 to 2023, which were set up in four sowing dates, including 10 d early sowing, 10 d late sowing, 20 d late sowing, and control. The growth process, above-ground dry matter accumulation and distribution, photosynthetic characteristics of leaves, grain filling rate, yield agronomic traits, and grain nutrients of winter wheat and summer maize were observed. 【Result】 The whole growth period of winter wheat was shortened with the delay of sowing date, mainly because of the shortening of seedling stage before winter. There was a parabolic relationship between the whole growth period and sowing date of summer maize. The seedling stage was shortened by 1.3 d, and the flowering stage and grain formation-filling stage were extended by 1.5 d and 1.6 d for every 10 d delay of sowing date. The grain filling characteristics of winter wheat and summer maize were not sensitive to sowing date adjustments, and the grain filling rate of summer maize was little different during different sowing dates, but the grain formation period, the filling end date and the peak date were successively delayed due to the delay of sowing date, and the duration of filling days was shortened by 4.0 d for every 10 d delay of sowing date. Under the background of warm autumn and winter in northern wheat region, the sowing duration of winter wheat was extended, while its influence on the yield was obviously weakened. The delay of sowing date with the increase of sowing seed amount would result in yield increase slightly. The yield of summer maize decreased significantly with the delay of sowing date, and the decline rate of theoretical yield was 1 381.50 kg·hm^-2 for every 10 d delay of sowing date, but the yield of winter wheat and summer maize showed a jumping decease for 20 d late sowing. The grain distribution rate increased by 1.67% for winter wheat, decreased by 1.57% for summer maize with every 10 d delay of sowing date. As a result, the harvest index increased by 0.017 for winter wheat, and decreased by 0.016 for summer maize with every 10 d delay of sowing date. The leaf photosynthetic rates (Pn) of winter wheat and summer maize were also different in response to sowing date, they were similar for winter wheat during different sowing dates, while decreased by 1.21 μmol·m^-2·s^-1 for summer maize for every 10 d delay after sowing date. Sowing date adjustments had no significant effects on grain quality of winter wheat and summer maize in northern area of North China. 【Conclusion】Extending suitable sowing date range and sowing date delay of winter wheat in North China Plain were positive and effective measures to adapt to climate warming. The early sowing of summer maize in North China Plain might avoid the negative effects of high temperature and heat damage, and would promote the increase of yield.

Screening and Verification of Pepper Host Factors Interacting with the 126 kDa Protein of Pepper Mild Mottle Virus by Yeast Two-Hybrid System

HE Yong, FAN XiaoZhu, CHEN XinYue, DUAN ShuJing, HU TingTing, XIE RuXue, WANG YuQing, CHEN Jing

Scientia Agricultura Sinica. 2024, 57(15): 2986-2996. doi:10.3864/j.issn.0578-1752.2024.15.006

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【Objective】Pepper mild mottle virus (PMMoV) is one of the major viruses that harm peppers worldwide. The 126 kDa protein is an important pathogenic factor encoded by PMMoV, but its pathogenic mechanism remains unclear. This study aims to screen for pepper host factors that interact with the 126 kDa protein, and to provide a theoretical basis for elucidating the pathogenic mechanism of PMMoV.【Method】Firstly, the bait vector pGBK-126 kDa was constructed using homologous recombination. Using pepper leaves as experimental materials, total RNA was extracted from pepper leaves using the Trizol method, and a pepper yeast cDNA library was prepared. Subsequently, the cDNA library was screened using pGBK-126 kDa, and the screening results were subjected to sequence alignment and bioinformatics functional analysis using NCBI and Uniprot. Based on the alignment and analysis results, host factors that may participate in plant disease resistance pathways were selected, and their full-length CDSs were cloned and constructed into the pGADT7 vector. Yeast two-hybrid (Y2H), BiFC, and LCI were used to further verify the interaction between 126 kDa and host factors. Finally, the role of transiently overexpressed host proteins during PMMoV infection was analyzed.【Result】High-quality pepper RNA was extracted without degradation. A high-quality yeast cDNA library was obtained, and the bait plasmid pGBK-126 kDa was successfully constructed. A total of 18 pepper host factors that interact with the 126 kDa protein were screened. Bioinformatics analysis revealed that these 18 host factors are widely involved in multiple pathways such as plant enzyme systems, regulation of material and energy metabolism, DNA-binding transcription, hormone synthesis, and defense responses. Among them, three host factors (LA2, PDHE1, BXL1) showed interactions with 126 kDa in one-to-one Y2H interaction verification, indicating the reliability of the initial screening results. The interaction between 126 kDa and BXL1 was further verified in vitro and in vivo using BiFC and LCI. Transient overexpression of BXL1 significantly inhibited PMMoV infection.【Conclusion】The pGBK-126 kDa bait plasmid was successfully constructed. Based on this plasmid, 18 interacting host factors were obtained when screening the yeast cDNA library, which are widely involved in multiple pathways of plant life activities. The screening results were verified to be reliable. Among them, BXL1 interacts with 126 kDa both in vitro and in vivo, and can inhibit PMMoV infection. The results can provide a good theoretical and material basis for further exploring of the infection mechanism of PMMoV.

Preparation of Fluopyram-Loaded Nanofiber Nematicide and Its Biological Activity Against Meloidogyne incognita

LING WenZheng, CAO HaiChao, YU JiaMin, ZONG Hao, WANG Kai, FENG Chao, CHEN Dan

Scientia Agricultura Sinica. 2024, 57(15): 2997-3009. doi:10.3864/j.issn.0578-1752.2024.15.007

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【Objective】Root-knot nematode disease has become an important influencing factor restricting the development of protected agriculture in China, among which Meloidogyne incognita has caused the most serious yield loss. At present, there are few types of nematicides in China, the objectives of this study are to prepare the fluopyram-loaded nanofiber nematicide based on the electrospinning technique, evaluate its biological activity against M. incognita, and to enrich the types of effective nematicides for controlling root-knot nematode disease of crops and expand the application field of nanofiber agents.【Method】Two kinds of fluopyram-loaded nanofiber nematicide (Flu@NF) were prepared by coaxial electrospinning technology with different biodegradable materials as shell materials and solvated fluopyram as core materials. The morphology, structure, elemental composition, functional components and thermal stability of Flu@NF were characterized by SEM, EDS, TEM, FTIR and TGA. With 41.7% fluropyram suspension as control agent, the biological activity of Flu@NF against 2nd stage juveniles (J2) and egg mass of M. incognita was evaluated.【Result】PEG/PCL-Flu@NF and CA-Flu@NF using polyethylene glycol (PEG), polycaprolactone (PCL) and cellulose acetate (CA) as shell materials were successfully prepared under high pressure +15 kV, core-shell solution flow rate 1﹕4 and receiver side distance of 24 cm. The average diameter of two kinds of agents was 316 and 440 nm, respectively. Fibers were in accordance with the inner and outer microstructure of core-shell, and the shell thickness was separately 71.15 and 24.84 nm. The loading capacity of two kinds of fluopyram-loaded fibers was 15.32% and 20.37%, respectively. Moreover, the thermal stability of fluopyram was significantly improved after shell coating by coaxial electrospinning. Compared with fluropyram suspension, the slow-release performance of fluopyram-loaded fibers was significantly improved, and the mortality of J2 was significantly increased with the extension of treatment time at different concentrations. The lethality rate of the two fiber agents against J2 was higher than 98% after 24 h treatment at high concentration (25 mg·L^-1). After treatment with low concentration (4 mg·L^-1) for 24 h, the lethality rate of the two fiber agents against J2 was 91.4% and 79.6%, respectively. The two kinds of nematicides had good inhibition effects on the hatching of nematode eggs, after 3 d treatment at high concentration (25 mg·L^-1), the unhatched rate of eggs was 71.8% and 89.2%, respectively, and after 3 d treatment at low concentration (4 mg·L^-1), the unhatched rate of eggs was 59.4% and 76.2%, respectively.【Conclusion】Based on coaxial electrospinning technology, the nanofibers prepared with PEG/PCL and CA as shell materials have excellent coating effect on fluopyram, and the fluopyram-loaded fibers have high biological activities on M. incognita through slow-release action, which has great potential to prevent and control root-knot nematode disease through crop root protection.

Effects of Soil Film Tillage on Soil and Crop in Farmland

YANG ShiQi, YAN Xin, HAN Yu

Scientia Agricultura Sinica. 2024, 57(15): 3010-3022. doi:10.3864/j.issn.0578-1752.2024.15.008

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【Objective】To make clear the agricultural productive value of soil film tillage, the effects of soil film tillage on soil moisture, temperature and nutrient changes in farmland were explored, and the effects of soil film on crop root growth and yield were revealed too, so as to provide the theoretical basis and technical methods for the application of soil film.【Method】Soil film was induced by spraying a 1.0% concentration of carboxymethyl cellulose ammonium (CMC-NH₄) aqueous solution, and the field experiment was conducted with five treatments, including 0 (CK), 50.0 (T1), 100.0 (T2), 200.0 (T3), and 300.0 kg·hm^-2 (T4). The effects of CMC-NH₄ application rate on soil moisture, temperature, nutrients, microorganisms, crop roots and yield in multiple cropping of spring wheat and summer maize were studied.【Result】Soil film treatment of spring wheat and summer maize could increase average daily soil moisture content by 3.3%-7.0% (P<0.05, the same as below) and 1.9%-6.1%, average daily temperature by 7.9%-12.6% and 5.6%-11.7%, contribution of soil accumulated temperature of growth period by 88.98-141.94 ℃ and 60.25-136.65 ℃, root length of 0-30 cm soil layer by 37.5%-17.1% and 11.2%-1.7%, root surface area of 0-30 cm soil layer by 15.3%-4.5% and 12.5%-9.2%, respectively, and root biomass (dry weight) in 0-30 cm soil layer were enhanced by 17.0%-41.5% and 30.9%-36.7%, respectively. Finally, the grain yield of spring wheat per unit area was increased by 7.3%-18.8% and above-ground dry weight of summer maize per unit area was increased by 33.6%-49.0%. and the soil nitrogen, phosphorus and potassium content as well as soil microbial diversity were improved too. 【Conclusion】As a novel type of soil tillage, soil film had the function of farmland coverage, which greatly contributed to improve soil hydrothermal environment, and promote nutrient absorption and utilization, root growth and crop yield. At the same time, soil film produced the important technique and method to make the better plough layer structure that is “compact surface, loose top and tight bottom of plough layer”, which would support the improvement of crop productivity, farmland soil amendment and agricultural high-quality development. In this study, the recommended application rate was 100.0 kg·hm^-2 in Ningxia Yellow River irrigation area.

Effects of Different Rice Varieties and Zinc Application Methods on Rice Yield and Grain Zinc Availability

SU SuMiao, KANG TianKai, ZOU JiaLong, WANG BenFu, ZHANG YangYang, LIAO ShiPeng, LI XiaoKun

Scientia Agricultura Sinica. 2024, 57(15): 3023-3034. doi:10.3864/j.issn.0578-1752.2024.15.009

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【Objective】The aim of this study was to clarify the response of different rice varieties to zinc fertilizer and the differences in the effects of different zinc fertilizer application methods on rice yield and grain zinc bioavailability.【Method】The field experiments were conducted in 2019 and 2020. The first field experiment (Experiment 1) in 2019 aimed to explore the response of different rice varieties to zinc fertilizer application. Four rice varieties were selected, including Xiadao No.1, Xiadao No.2, Runxiangyu, and Longliangyou3463, with two treatments of no zinc application (CK) and zinc application (+Zn), and the differences in the response of different rice varieties to zinc fertilizer application were investigated. The second field experiment (Experiment 2) in 2020 aimed to explore the effects of different zinc fertilizer application methods on rice yield and grain zinc bioavailability. Based on the experiment in 2019, two high-yielding rice varieties, including Xiadao No.1 and Longliangyou3463, were selected to continue the study. Six treatments were set: no zinc application to soil (S0F0), soil application of 15 kg·hm^-2 zinc (S1F0), soil application of 30 kg·hm^-2 zinc (S2F0), foliar spray of 0.5% ZnSO₄ at flowering stage (S0F1), soil application of 15 kg·hm^-2 zinc + foliar spray of 0.5% ZnSO₄ at flowering stage (S1F1), and soil application of 30 kg·hm^-2 zinc + foliar spray of 0.5% ZnSO₄ at flowering stage (S2F1), and the differences in the effects of different zinc application methods on yield and grain zinc bioavailability of two rice varieties were investigated.【Result】In experiment 1, there were significant differences in the response of different rice varieties to zinc fertilizer. After zinc application, the yields of Xiadao No.1, Xiadao No.2, Runxiangyu, and Longliangyou3463 increased by 4.4%, 11.6%, 7.9%, and 4.8%, respectively. The increase in yield was mainly attributed to the increase in effective panicle number and grain-setting rate. Compared with CK, the average effective panicle number of the four rice varieties with zinc application increased by 19.8%, 3.9%, 9.9%, and 24.0%, respectively, and the average grain-setting rate increased by 5.4%, 9.7%, 6.7%, and 4.0%, respectively. In addition, the molar ratio of phytic acid to zinc in brown rice and polished rice of the four rice varieties with zinc application decreased by averages of 24.3%, 30.9%, 21.3%, and 37.2%, and 29.5%, 32.4%, 33.9%, and 35.3%, respectively. In experiment 2, there were significant differences in the effects of different zinc application methods on the yield and grain zinc availability of Xiadao No.1 and Longliangyou3463. Soil zinc application significantly outperformed foliar zinc spray in increasing yield, and foliar zinc spray further increased rice yield when it combined with soil zinc application. Compared with S0F0, the yields of Xiadao No.1 under S0F1, S1F0, S1F1, S2F0, and S2F1 increased by 12.1%, 14.1%, 17.2%, 22.2%, and 29.3%, respectively, and the yields of Longliangyou3463 under S0F1, S1F0, S1F1, S2F0, and S2F1 increased by 2.0%, 10.1%, 15.2%, 28.3%, and 31.3%, respectively. The effects of different zinc application methods on grain zinc availability varied, and there were significant differences in grain zinc availability between the two rice varieties. Foliar spray had a significantly greater zinc enhancement effect on grain compared with soil application, and the bioavailable zinc content in grains of Longliangyou3463 was higher than that in Xiadao No.1.【Conclusion】Selecting high-yielding and zinc-enriched rice varieties (Longliangyou3463) and adopting appropriate zinc fertilizer application methods(30 kg·hm^-2 soil application + foliar spray of 0.5% ZnSO₄ at flowering stage) could achieve the goals of increasing rice yield and enhancing rice zinc nutrition, thereby helping to increase income and improve rice quality.

Transcriptome Analysis of Paeonia lactiflora in Response to Alternaria tenuissima Infection

YU ShuHan, QIN XiaoJie, WU QiChao, LI Ling, ZANG DeKui, MA Yan

Scientia Agricultura Sinica. 2024, 57(15): 3035-3052. doi:10.3864/j.issn.0578-1752.2024.15.010

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【Objective】Paeonia lactiflora (P. lactiflora) is prone to pathogen infection during its growth and development. Leaf red spot disease caused by the Alternaria tenuissima (A.tenuissima) seriously affects both the quality and yield of the plant. However, the disease resistance mechanism of P. lactiflora is not clear at present. This study aimed to explore the physiological changes and molecular response pathways of P. lactiflora in response to the infection of A. tenuissima by using physiological and transcriptomic approaches.【Method】Taking P. lactiflora Dafugui as the experimental material, the leaves of them at 12, 24 and 96 h after A.tenuissima infection were taken respectively to determine the relevant physiological indexes and perform transcriptome sequencing analysis, used uninoculated leaves as the control.【Result】After pathogen inoculation, the activities of SOD, POD, CAT and PAL of the leaves of P. lactiflora increased, the content of soluble sugar, soluble protein and MDA increased, but the content of proline decreased. The differentially expressed genes (DEGs) in each stage were screened after 12, 24 and 96 h of A. tenuissima infection. There were 5 045, 5 961 and 2 748 DEGs up-regulated, and 4 284, 5 665 and 3 536 DEGs were down-regulated in each stage. GO enrichment analysis showed that these DEGs were mainly enriched in some biosynthesis and metabolic processes, photosynthesis-related, signal transduction-related and rhythm-related items. KEGG enrichment analysis showed that these DEGs were mainly enriched in carbon metabolism, amino acid biosynthesis, plant-pathogen interaction, plant hormone signal transduction, MAPK signaling, and other pathways. A total of 53 DEGs were enriched in three disease resistance pathways (plant-pathogen interaction, MAPK signaling and plant hormone signal transduction pathways). These DEGs included 1 MPK6, 2 PR1, 4 MKK4/5, and 46 BAK1. Nine differentially expressed genes of P. lactiflora in response to A. tenuissima infection were selected for qRT-PCR analysis. The gene expression pattern was consistent with the transcriptome sequencing results, which confirmed the accuracy of RNA-seq. The transcription factor families analysis showed that the AP2/ERF-ERF, WRKY, bHLH and MYB-related transcription factor families were the key transcription factor families of P. lactiflora in response to A.tenuissima.【Conclusion】After A.tenuissima infected, the antioxidant capacity of P. lactiflora was increased by increasing the activities of SOD, POD, CAT, and PAL, to remove the large amount of reactive oxygen species produced by disease stress, maintaining cell water by increasing the content of soluble sugars and soluble proteins and reducing the content of proline. The continuous increase of MDA content indicated that the cell membrane structure of plants was destroyed. The BAK1, PR1, MKK4/5 and MPK6 genes played important roles in response to A. tenuissima infection. It was preliminarily speculated that PlERF20, PlERF1b, PlWRKY41, PlMYC4, and PlMYB62 were disease resistance related transcription factor in response to A. tenuissima infection.

Identification of Key Genes in the Flavonoid Synthesis Pathway of Meconopsis integrifolia Based on WGCNA

CHEN XiaoJuan, WANG HaiJu, WANG FuMin, YONG QingQing, HUANG ShunMan, QU Yan

Scientia Agricultura Sinica. 2024, 57(15): 3053-3070. doi:10.3864/j.issn.0578-1752.2024.15.011

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【Objective】Flavonoids are known for their anti-inflammatory, anti-cancer, and antibacterial properties, and are one of the main medicinal components of Meconopsis integrifolia. By analyzing the spatial metabolome information and transcriptome data from various parts (roots, stems, leaves and petals) of M. integrifolia, the key genes regulating the flavonoid synthesis could be excavated. This research could provide valuable insights into the mechanism underlying flavonoids synthesis in M. integrifolia, paving the way for genetic breeding aimed at enhancing flavonoid content. 【Method】Transcriptome sequencing was conducted on the root, stem, leave and petal of M. integrifolia to analyze the distribution of flavonoids across different parts using spatial metabolomic data. The weighted gene co-expression network analysis (WGCNA) was employed to identify key modules and key genes closely related to flavonoid synthesis. To validate the reliability of the transcriptome data, 12 genes were selected for qRT-PCR analysis. 【Result】Flavonoids accumulation in M. integrifolia varied across different parts, with petals being the primary site of accumulation, where 8 main flavonoids were identified. Transcriptome sequencing revealed a total of 20 085 expressed genes, among which 286 genes expressed were exclusively expressed in flowers, showing 3.6-4.2 times more expression than that in other plant parts. Using WGCNA to categorize highly expressed differential genes, a total of 14 co-expression modules were identified, and the key modules, including MEturquoise and MEgreen, were significantly associated with 8 main flavonoids (P<0.05). KEGG analysis demonstrated that the genes within these two modules were primarily enriched in metabolism-related pathways, with some genes enriched in pathways related to flavonoid synthesis. MEturquoise and MEgreen comprised 18 and 6 genes related to flavonoid synthesis, respectively, and screened 14 core structural genes (5 CHSs, 2 HIDHs, 2 CCoAOMTs and FLS, CYP75B1, CHI, HCT, and CYP73A) and one transcription factor HB2, which were predominantly highly expressed in petals or stems. The consistent gene expression trends between qRT-PCR and transcriptome data were observed, which showed that the analysis results derived from the transcriptome data were reliable. 【Conclusion】The accumulation of flavonoids and gene expression patterns in different organs of M. integrifolia varied significantly, and 14 core structural genes and one transcription factor were screened to be closely related to the accumulation of flavonoids across different organs. These genes might play a key role in regulating the synthesis and differential accumulation of flavonoids in different organs of M. integrifolia.

Effects of the Different Tomato Addition Ratios on the Eating Quality and Oxidation of Stewed Beef

YOU Min, CHEN JinSong, CHEN YanYan, YANG Ping, ZHANG ChunHui, HUANG Feng

Scientia Agricultura Sinica. 2024, 57(15): 3071-3082. doi:10.3864/j.issn.0578-1752.2024.15.012

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【Objective】This study aimed to clarify the effects of adding tomatoes on the quality characteristics, lipid and protein oxidation inhibition of stewed beef, so as to provide insights into the interaction mechanism between tomato and beef components, thereby contributing to the development of a standardized production process.【Method】Tomato was added to the stewed beef in proportions of 0, 50%, 100%, 150%, and 200%, respectively, and which were stewed for 20 min. The quality characteristics of stewed beef, including pH value, color, and tenderness, were measured. The content of amino acids, nucleotides, and the taste of stewed beef brisket with tomato were determined and evaluated. Besides, the degrees of lipid and protein oxidation in beef were studied as well. 【Result】With higher ratios of added tomato, the pH value and L* value of beef significantly decreased (P<0.05), while the a* value increased. On the other hand, the shear force of beef significantly decreased and the myofibrillar fragmentation index (MFI) increased (P<0.05), which positively affected the color and tenderness of beef. Higher content of aspartic acid, glutamic acid, and proline in the soup and higher content of free amino acids such as glutamic acid and histidine in beef were observed as tomato added, while the content of 5′-AMP, 5′-GMP and 5′-IMP in beef were decreased (P<0.05). The addition of tomato resulted in a reduction in the levels of beef Schiff base (SB), TBARS, and the total sulfhydryl group. However, it increased the free sulfhydryl content. There was no significant effect on the carbonyl content. It could be assumed that tomatoes could inhibit the lipid oxidation and protein oxidation of stewed beef. The tomato addition ratios of 100%, 150%, and 200% had no significant effect on sensory acceptability. However, combining the content of free amino acids, nucleotides and the lipid and protein oxidation of beef, it was found that adding 200% tomato during beef stew resulted in better edible quality and greater oxidation inhibition ability. 【Conclusion】The addition of tomato stew could increase the redness, tenderness, and free amino acid content, and accelerate the release nucleotides of beef. Addition of tomatoes could inhibit the lipids oxidation and proteins oxidation of beef. Overall, addition of tomatoes results in beef with good overall acceptability and suppress oxidation.

Mechanism of miRNA-424-5p Regulate the Decomposition of Collagen on Retained Fetal Membranes of Dairy Cows Through VEGFA Pathway

LIU BingQi, LUO ChunHai, YAO WeiJia, WANG Wei, LIU JiaJin, LI DanYang, FU ShiXin

Scientia Agricultura Sinica. 2024, 57(15): 3083-3092. doi:10.3864/j.issn.0578-1752.2024.15.013

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【Objective】To investigate the effect of miRNA-424-5p on collagen degradation in placental tissues of dairy cows, and then to clarify the regulatory role and mechanism of miRNA-424-5p on the occurrence of retained fetal membranes (RFM) in dairy cows.【Method】The expression level of miRNA-424-5p in maternal placenta tissues of normal cows and cows with RFM were firstly detected, and the expression levels of VEGFA, MMP-2, MMP-9 and COL-IV in maternal placenta tissues of normal cows and cows with RFM were detected by qRT-PCR and Western blot. Prediction of target genes for miRNA-424-5p was performed using bioinformatics analysis, the targeting relationship of miRNA-424-5p with VEGFA was verified using the dual luciferase assay. miRNA-424-5p mimics and miRNA-424-5p inhibitor were transfected in dairy endometrial epithelial cells for overexpression and silencing of miRNA-424-5p. Immunofluorescence was used to observe the changes of VEGFA expression in dairy endometrial epithelial cells. The expression of VEGFA, MMP-2, MMP-9 and COL-IV were detected by qRT-PCR and Western blot.【Result】Compared with the healthy group, miRNA-424-5p mRNA expression level in maternal placental tissues of RFM group was significantly increased (P<0.01), the expression of VEGFA, MMP-2, MMP-9 mRNA and protein was highly significant decreased (P<0.01). COL-IV mRNA and protein was highly significant increased (P<0.01). There were 152 potential target genes of miRNA-424-5p, and VEGFA was confirmed to be the target gene of miRNA-424-5p by the results of dual luciferase assay.After overexpression and silencing of miRNA-424-5p, the immunofluorescence results showed that the expression of VEGFA was lower in the miRNA424-5p mimics group compared with the control group, while the expression of VEGFA was higher in the miRNA424-5p inhibitor group. The qRT-PCR and Western blot results showed that overexpression of miRNA-424-5p, the expression of target gene VEGFA mRNA and protein were significantly decreased, and the expression of MMP-2 and MMP-9 mRNA and protein were highly significantly down-regulated (P<0.01), and COL-IV mRNA and protein expression levels were highly significantly increased (P<0.01).【Conclusion】miRNA-424-5p can through VEGFA pathway to regulate the degradation of MMPs and collagen, and can cause the obstacle of extracellular collagen degradation, miRNA-424-5p is an important factor of causing the occurrence of retained fetal membranes.

Antigenic Variation of Foot-and-Mouth Disease Virus Serotype O in Southeast Asia Topotype

WANG Li, LI Kun, HUANG ShuLun, LI FengJuan, WANG Sheng, LU ZengJun, LIU ZaiXin

Scientia Agricultura Sinica. 2024, 57(15): 3093-3104. doi:10.3864/j.issn.0578-1752.2024.15.014

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【Objective】The continuous variation of foot-and-mouth disease virus (FMDV) serotype O easily leads to the decrease of antigen matching between the currently used vaccine and the circulating strain, resulting in poor immune effect and sporadic outbreaks. In recent years, Southeast Asia (SEA) topotype epidemic of FMDV serotype O has been active and constantly changing, which has continued to exert pressure on foot-and-mouth disease prevention. Systematic analysis of specific antigen sites of serotype O FMDV strains and sequence differences of different topological strains and identification of key amino acids of antigen variation could provide basis for FMD antigen molecular design, and provide guidance for FMD prevention and control. 【Method】In this study, 10 bovine-derived monoclonal neutralizing antibodies specific for SEA topotype strains were used to screen escape mutants by antibody pressure in strain O/GSLX/2010 (O/Mya/98 lineage) to identify the key amino acids. The viral cross-neutralization tests were performed using bovine polyclonal antiserum samples (32 samples) and escape mutants to analyze the immunodominant epitopes of SEA topotype strains. The differences in this epitope between different topotype strains were analyzed by sequence alignment. Reverse genetic technique was used to introduce differential epitopes into FMDV serotype O classic vaccine strain O/HN/CHA/93 (Cathay topotype), and the rescued point mutant strains were sequenced and identified by indirect immunofluorescence assay. Virus plaque detection and one-step growth curve were used to detect the replication dynamics of the virus. Using SEA topotype strain specific monoclonal neutralization antibodies, the antigenicity of the rescue mutant was analyzed by neutralization test, the key amino acids affecting the antigenicity of the virus were identified, and the influence of the amino acids changes at the specific site of the strain on the antigen profile was evaluated. 【Result】SEA topotype-specific epitopes were mainly concentrated in the B-C and C-D loop of VP1, which belonged to antigenic site 3. Most of the 10 mAbs (8/10) recognized epitopes on VP1, among which 6 mAbs (A19, B55, B74, C5, F53, and F166) recognized key amino acids located in the B-C loop (T43, K45) and C-D loop (P58) of VP1, and the other 2 mAbs (B66 and F41) identified key amino acids at the C-terminus of VP1. The results of virus cross-neutralization test showed that the changes of amino acids 43 of VP1 and 131 of VP3 significantly reduced the antibody titer of bovine polyclonal antisera. The sequence analysis of different lineages of FMDV serotype O (26 strains) revealed that amino acids 28, 47, 56, and 58 in the B-C/C-D loop of VP1 were different in the three topotype. Using reverse genetic technology, the virus-specific epitope was successfully introduced into Cathay topotype (O/HN/CHA/93) and saved 6 FMDV point mutants: POZ-GSLX-M58, POZ-GSLX-M56/58, POZ-GSLX-M28/58, POZ-GSLX-M47/58, POZ-GSLX-M28/58/47, and POZ-GSLX-M47/56/58. The results of microneutralization test showed that 56/58 VP1 played a key role in the antigenicity of the virus. However, further additions of mutations at positions 28 and 47 reduced the neutralization of antibody B83, affecting the antigenicity of the strain itself. Therefore, the changes in amino acids at positions 56 and 58 on VP1 could extend the effectiveness and breadth of SEA topotype specific neutralization of mAbs against O/HN/CHA/93 strains.【Conclusion】Amino acids 56 and 58 on the structural protein VP1 of FMDV serotype O were key sites causing antigenic variation in SEA topotype, and introducing these antigenic determinants could expand the neutralization potency and breadth of SEA topotype-specific neutralizing mAbs, and effectively expand the antigenic profile of FMDV O/HN/CHA/93. This study provided an important reference for FMD prevention and vaccine design.

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