Scientia Agricultura Sinica

QTL Identification and Genetic Analysis of Plant Height in Wheat Based on 16K SNP Array

YAO QiFu, CHEN HuangXin, ZHOU JieGuang, MA RuiYing, DENG Liang, TAN ChenXinYu, SONG JingHan, LÜ JiJuan, MA Jian

Scientia Agricultura Sinica. 2023, 56(12): 2237-2248. doi:10.3864/j.issn.0578-1752.2023.12.001

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【Objective】There is a close relationship between plant height (PH) and yield. The aim of this study is to further explore quantitative trait loci (QTL) of PH with breeding value in wheat and analyze the genetic effects of major QTL for PH on other yield related traits toward to providing a theoretical basis for molecular breeding. 【Method】A recombinant inbred line population (MC) derived from a cross between the natural mutant msf and Chuannong 16 (CN16) was used for QTL analysis. During 2020 to 2022, planting and PH phenotype identification were conducted at five environments in Wenjiang, Chongzhou, and Ya’an of Sichuan Province. The high-quality genetic linkage map constructed using the 16K SNP array was used for QTL mapping of PH. Genotypes of flanking markers of major QTL for PH were used to analyze the genetic effects of positive alleles on yield related traits and evaluate the potentiality of QTL for yield improvement. 【Result】Eight QTL controlling PH were identified on chromosomes 1A, 3D, 4D, 5A, and 7B, respectively. Among them, two stable and major QTL, QPh.sau-MC-1A and QPh.sau-MC-5A, were located, which explained 9.09% to 25.56% and 3.91% to 13.09% of the phenotypic variation rate, respectively. Their positive alleles were all from CN16. The additive effect analysis showed that PH of the lines carrying positive alleles from QPh.sau-MC-1A and QPh.sau-MC-5A was significantly higher than that of the lines carrying only a single positive allele or none. Correlation analysis showed that PH has a significantly positive correlation with effective tiller number (ETN), a significantly negative correlation with flag leaf width (FLW), and no significant correlation with kernel number per spike (KNPS), kernel weight per spike (KWPS), thousand kernel weight (TKW), flag leaf length (FLL) and anthesis date (AD). Genetic effects analysis showed that positive allele of QPh.sau-MC-1A had a significant effect on improving ETN (56.51%), a significant effect on decreasing KNPS (-11.26%), KWPS (-13.04%), TKW (-5.47%), and FLW (-2.85%), and a significant effect on advancing AD (-0.61%). Positive allele of QPh.sau-MC-5A had a significant effect on improving ETN (10.57%), KNPS (4.32%), and TKW (2.92%), and a significant effect on delaying AD (1.07%). 【Conclusion】A major QTL QPh.sau-MC-5A for PH was mapped on chromosome 5A, and its positive allele significantly increased ETN, KNPS, and TKW, indicating that it may have a positive impact on yield.

Construction of DNA Molecular Identity Card of Core Germplasm of Broomcorn Millet in China Based on Fluorescence SSR

XUE YaPeng, DING YiBing, WANG YuZhuo, WANG XiaoDan, CAO XiaoNing, SANTRA Dipak K, CHEN Ling, QIAO ZhiJun, WANG RuiYun

Scientia Agricultura Sinica. 2023, 56(12): 2249-2261. doi:10.3864/j.issn.0578-1752.2023.12.002

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【Objective】As an ancient minor grain crop, broomcorn millet ( Panicum miliaceum L. ) is abundant in germplasm. The construction of their DNA molecular identity based on fluorescent SSR markers would provide theoretical basis and molecular detection tool for digital management of resources. 【Method】Two hundred and thirty five broomcorn millet core accessions from China were used as experimental material, polymerase chain reaction were conducted several times using the broomcorn millet specific SSR markers which developed previously by the Broomcorn Millet Crop Molecular Breeding Research Group of the Agronomy College in Shanxi Agricultural University, core markers were obtained. With the given reference genome information of broomcorn millet, the core markers were mapped on chromosomes through BLAST sequence alignment. Fluorescence (FAM/HEX) was labeled on the 5' end of the SSR primer, the genotype of the material was given by capillary electrophoresis. Using binary coding means of expression, “0, 1” was written representing the presence or absence of amplified bands, and the discrimination of the material was detected by the software ID Analysis 4.0. Decimal (0-9) coding methods were used to calculate the size of the amplified fragments so as to obtain the character string molecular identity card of the accession. Genetic diversity, genetic clustering and principal component analysis were performed using the softwares Popgene, Powermarker, MEGA and NTSYS. The two-dimensional code DNA molecular identity card of the accession was given using the two-dimensional code online software (https://cli.im/). 【Result】PCR amplification results showed that all the 235 accessions could be separated by 7 fluorescent SSR markers (RYW3, RYW6, RYW11, RYW18, RYW37, RYW43 and RYW125) combined together. BLAST results showed that RYW18 and RYW37 were distributed on Chromosome 2, located at 0.60 cM and 0.80 cM, respectively. RYW125 is located on Chromosome 4 at 10.40 cM. RYW43 and RYW6 were distributed on Chromosome 5, located at 52.80 cM and 53.00 cM, respectively. RYW11 and RYW3 were located on Chromosome 6 at 2.10 cM and 20.70 cM, respectively. Genetic diversity analysis showed that 87 alleles were detected at 7 loci among all accessions, 3 (RYW11)-25 (RYW6) alleles were detected at each locus, with an average of 12.4286. Shannon diversity index (I) was detected and ranged from 0.2055 (RYW18) to 2.0587 (RYW6), with an average of 1.1398. The observed heterozygosity (Ho) was 0.0086 (RYW11)-0.9455 (RYW18). The expected observed heterozygosity (He) was 0.0795 (RYW18)-0.7469 (RYW11). Nei’s gene diversity index (Nei) was 0.0793 (RYW18)-0.7452 (RYW6). The polymorphism information content (PIC) was 0.0334 (RYW11)-0.8071 (RYW6), with an average of 0.5185. The results of cluster analysis and principal component analysis showed that 235 accessions were classified into 8 groups. The electrophoretic bands were number coding, and 7 marker combinations were used to construct the character string and two-dimensional code DNA molecular ID of all the accessions.【Conclusion】Two hundred and thirty five broomcorn millet core germplasms from China were used as material, polymerase chain reaction and capillary electrophoresis were conducted, 7 core SSR markers were screened. With the given reference genome information of broomcorn millet, the above markers were mapped on 4 chromosomes. Used the above SSR markers, genetic diversity analysis of all accessions was conducted and genetic diversity parameters were obtained. Based on Cluster analysis, all accessions were classified into 8 groups. Principal component analysis result resolved the deviation occured in Cluster analysis. According to the principle of most accessions were tell apart using the least markers, decimal (0-9) coding methods were used to calculate the size of the amplified fragments so as to obtain the character string molecular identity card of the accession. Combined the phenotype data with the above character string, two-dimensional code DNA molecular ID of all the accessions were developed.

Effects of Cultivation Modes on Soil Physicochemical Properties and Nitrogen Balance in Wheat Fields Under Long-Term Positioning Conditions

GUO XinHu, MA Jing, LI ZhongFeng, CHU JinPeng, XU HaiCheng, JIA DianYong, DAI XingLong, HE MingRong

Scientia Agricultura Sinica. 2023, 56(12): 2262-2273. doi:10.3864/j.issn.0578-1752.2023.12.003

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【Objective】 From the 2009-2010 wheat growing season, four cultivation modes were designed and set up. The effects of cultivation modes on soil physical and chemical properties, nitrogen nutrition index of winter wheat, nitrogen supply and demand balance in wheat field, uptake and utilization of nitrogen and grain yield were investigated, in order to provide a theoretical guidance for further optimizing the soil-crop system integrated management mode.【Method】Four cultivation modes were designed: local farmer mode (T1), improvement mode based on farmers (T2), high-yield and higher-yield mode regardless of production cost (T3), and soil-crop system integrated management mode (T4).【Result】After 13 wheat-maize growing seasons, the soil bulk density of surface soil for T1, T2, T3 and T4 modes decreased by 6.21%, 9.80%, 12.25% and 13.56%, respectively; the content of organic matter for four modes increased by 21.88%, 26.80%, 32.05% and 36.39%, respectively; the corresponding increases were 34.16%, 12.38%, 39.60% and 20.79% for the contents of total nitrogen; 47.85%, 48.87%, 74.49% and 62.21% for the contents of alkali-hydrolysable nitrogen, respectively; 62.73%, 36.56%, 297.93% and 68.68% for the contents of available phosphorus; 14.36%, 40.00%, 221.20% and 59.60% for the contents of available potassium, respectively. The increases of 33.96%, 10.32%, 52.77% and 19.49% were observed in the inorganic nitrogen accumulation in the 0-100 cm soil layer, respectively. Correspondingly, the pH for T1, T2, T3 and T4 modes decreased from 7.50 to 6.28, 6.68, 5.35 and 6.64, respectively. There were significant differences in grain yield and nitrogen uptake and utilization among the four cultivation modes in 2020-2022 growing season. Compared with T1 mode, the grain yield of T2, T3 and T4 modes increased by 14.14%, 27.65% and 22.52%, respectively; the nitrogen use efficiency increased by 54.80%, 19.97% and 49.15%, respectively; the nitrogen recovery efficiency increased by 72.95%, 37.54% and 48.15%, respectively; the nitrogen surplus decreased by 49.76%, 11.62% and 44.14%, respectively; the nitrogen surplus rate decreased by 24.63%, 11.62% and 26.68%, respectively. The whole plant at anthesis stage and spikes at maturity stage under T4 mode were in nitrogen supply and demand balance.【Conclusion】After 13 wheat-maize growing seasons, the soil acidification trend of 0-20 cm was obvious, and the bulk density of surface soil decreased, but the contents of organic matter, total nitrogen and available nutrients such as nitrogen, phosphorus, potassium increased for the all four cultivation modes. Meanwhile, the accumulation of inorganic nitrogen in 0-100 cm soil layer increased accordingly. Compared with other three cultivation modes, a synergistic improvement was obtained under T4 mode in soil physicochemical properties, wheat grain yield and nitrogen use efficiency. However, the nitrogen use efficiency at present under T4 mode was not high enough and still needed to be further improved. As showed by present study, further synergistic optimization in grain yield and nitrogen use efficiency could not be achieved only by reducing nitrogen input.

Optimization of Dryland Wheat Grain Growth Model Parameters Based on an Improved Shuffled Frog Leaping Algorithm

CUI WeiNan, NIE ZhiGang, LI Guang, WANG Jun

Scientia Agricultura Sinica. 2023, 56(12): 2274-2287. doi:10.3864/j.issn.0578-1752.2023.12.004

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【Objective】As the core decision module for intelligent agricultural production, the accurate simulation of the crop model depends on efficient and accurate optimization of the model parameters. In order to improve the efficiency of tuning parameters and enhance the performance and accuracy of the crop model, this study optimized the single objective parameters of the dryland spring wheat grain growth sub-model by improving the optimization algorithm, so as to provide a reference for the adaptation study of dryland spring wheat in the loess hilly region of northwestern China, to expand the application of the model, and to facilitate the model to better guide agricultural production.【Method】Based on a field experiment in Anjiapo Village, Fengxiang Town, Anding District, Dingxi City, Gansu Province, from 2015 to 2021, this study combined weather data and yearbook yield data from 1970 to 2021, further optimized six parameters of dryland wheat grain growth stage using roulette selection strategy based on the global communication and local depth search of traditional shuffled frog leaping algorithm (SFLA), carried out error calculation and comparison between measured and simulated yield values before and after algorithm improvement, and tested the APSIM-Wheat model.【Result】(1) At the same number of iterations, the traditional shuffled frog leaping algorithm converged around 200 times, while the improved shuffled frog leaping algorithm converged around 100 times. (2) The optimized parameters for the dryland spring wheat grain growth stage were: the grain number per gram stem was 26.0; the potential rate of grain filling from flowering to start of grain filling period was 0.00119 grain/d; the potential rate of grain filling during grain filling period was 0.00174 grain/d; the potential rate of grain filling under N limitation was 6.20×10^-5 g grain/d; the minimum rate of grain filling under N limitation was 1.90×10^-5 g grain/d; the maximum grain dry weight per plant was 0.0437 g. (3) The wheat yield was simulated using the parameter values optimized by the traditional shuffled frog leaping algorithm and the parameter values optimized by the improved shuffled frog leaping algorithm, respectively. After parameter optimization, the root mean square error (RMSE) between the measured and simulated yield values decreased from 363.22 kg·hm^-2 to 57.85 kg·hm^-2, and the normalized root mean square error (NRMSE) decreased from 21.78% to 3.47%.【Conclusion】Compared with the traditional shuffled frog leaping algorithm, the improved shuffled frog leaping algorithm increased the diversity of populations and subpopulations, converged quickly, and improved the optimization efficiency and accuracy, so the optimized results conformed to the growth and development process of dryland spring wheat with higher applicability, which significantly improved the performance of the APSIM-Wheat model in the loess hilly agricultural area of northwestern China.

Development of a Degenerate Primer RT-PCR Assay for Detection of Carmovirus

LIAO FuRong, CHEN HongYun, SHEN JianGuo, FANG ZhiPeng, HUANG PengYing, CHEN Qing, LIN LingLing, HONG Jun

Scientia Agricultura Sinica. 2023, 56(12): 2288-2301. doi:10.3864/j.issn.0578-1752.2023.12.005

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【Objective】The objective of this study is to establish a rapid, sensitive, and broad-spectrum screening method for simultaneous detection and identification of the genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus using degenerate primer RT-PCR combined with sequence analysis.【Method】Multiplexed analysis of genome sequences was aligned to search for suitable conserved regions for the design of the degenerate primers. One pair of degenerate primer Carmo-F2/Carmo-R2 was designed based on the RNA-dependent RNA polymerase (RdRp) gene sequences, and another pair primer Carmo-F2a/Carmo-R2a was formed by adding the non-complementary AT-rich sequences (AATAAATCATAA) to the 5′ end of the degenerate primers. The broad-spectrum, specificity, and sensitivity of RT-PCR method were analyzed. The sequencing, BLASTn analysis and phylogenetic analysis of PCR products were performed. The method was used to screen and detect viruses on Chinese hibiscus (Hibiscus rosa-sinensis) samples from Xiamen, China.【Result】Degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a were used to amplify partial RdRp gene of the members of genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus by RT-PCR. The fragment of approximately 500 and 550 bp was amplified, respectively. The developed RT-PCR assay was successfully used to detect angelonia flower break virus (AnFBV; Alphacarmovirus), calibrachoa mottle virus (CbMV; Alphacarmovirus), carnation mottle virus (CarMV; Alphacarmovirus), and pelargonium flower break virus (PFBV; Alphacarmovirus), hibiscus chlorotic ringspot virus (HCRSV; Betacarmovirus), melon necrotic spot virus (MNSV; Gammacarmovirus). The specificity test showed that no specific band could be obtained from maize chlorotic mottle virus (MCMV; Machlomovirus), pelargonium line pattern virus (PLPV; Pelarspovirus), carnation ringspot virus (CRSV; Dianthovirus), and healthy plants which including watermelon, melon, pumpkin, soybean, and pea. The sensitivity results showed that the primers Carmo-F2/Carmo-R2 could be detected up to 10^-2 dilution and the primers Carmo-F2a/Carmo-R2a could be detected up to 10^-3 dilution, which indicated that the non-complementary AT-rich sequences added at the 5′ end of the degenerate primers could increase the sensitivity of degenerate primer RT-PCR. BLAST analysis showed that the sequences determined had the highest sequence consistency with the corresponding virus species. Phylogenetic analysis based on partial amino acid sequences of the RdRp gene showed that it is consistent with the current classification of the subfamily Procedovirinae, and the viruses could be identified at the species level. HCRSV was detected in all 13 H. rosa-sinensis samples with suspected virus symptoms.【Conclusion】The RT-PCR method based on the degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a can be used for the screening and detection of viruses of the genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus, and can be used for rapid identification of virus species in combination with sequence analysis and phylogenetic analysis, and may be used to discover the new virus. H. rosa-sinensis plants were infested with HCRSV in Xiamen, China.

Expression and Ligand Binding Characteristics of GfunOBP2 from Grapholita funebrana

NIAN HeFen, ZHANG YuXi, LI BoLiao, CHEN XiuLin, LUO Kun, LI GuangWei

Scientia Agricultura Sinica. 2023, 56(12): 2302-2316. doi:10.3864/j.issn.0578-1752.2023.12.006

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【Objective】The objective of this study is to determine the binding affinities of the Plus-C odorant binding protein 2 of Grapholita funebrana (GfunOBP2) to sex pheromones and volatile compounds from apple trees, and to provide a basis for explaining the olfactory molecular mechanism of locating the host plants of G. funebrana.【Method】The ORF of GfunOBP2 was cloned by RT-PCR, and GfunOBP2 was identified as a Plus-C OBP subfamily protein through homology annotation and alignment of cysteine distribution patterns in amino acid sequences. The relative expression level of GfunOBP2 in the antenna, head, thorax, leg, wing, abdomen, and sex gland of the 3-day-old adults of G. funebrana was detected by RT-qPCR. The prokaryotic expression vector pET30a(+)/GfunOBP2 was constructed, and the recombinant GfunOBP2 protein was expressed in Escherichia coli BL21 (DE3) cells. The binding affinity of recombinant GfunOBP2 protein to five sex pheromones and 35 plant volatiles of apple trees was determined by using a fluorescence competitive binding assay. The interaction force and key amino acid residues of GfunOBP2 interacting with odorant ligands with strong binding affinities were predicted by molecular docking.【Result】The full-length ORF sequence of GfunOBP2 (GenBank number: OQ054799.1) was cloned, encoding 183 amino acids. It was found that GfunOBP2 has 12 conserved cysteines, and the distribution motif of cysteine residues indicated that GfunOBP2 belongs to the Plus-C OBP subfamily. GfunOBP2 was mainly expressed in the antennae of adults, and the relative expression level in male antennae was significantly higher than that in female antennae (P<0.05). Recombinant GfunOBP2 protein exhibited strong binding affinities to (E)-2-hexen-1-ol, benzyl alcohol, 1-heptanol, 1-decanol, hexanal, heptanal, cis-3-hexenyl acetate, cis-3-hexenyl 2-methylbutanoate, α-ocimene, β-caryophyllene, α-pinene and limonene, and the inhibition constant (K_i) for each ligand above was less than 5.0 μmol·L^-1. The molecular docking results showed that hydrogen bonds, donor-donor interactions, and alkyl interactions are the main weak interactions between GfunOBP2 and (E)-2-hexen-1-ol, 1-heptanol, and 1-decanol. The conventional hydrogen bonds and carbon hydrogen bonds are the main weak interactions between GfunOBP2 and cis-3-hexenyl acetate and cis-3-hexenyl 2-methylbutanoate. The alkyl interaction is the only weak force of GfunOBP2 interacting with α-ocimene and β-caryophyllene. Several hydrophobic amino acid residues, including Ile, Pro, Phe, Ala, Leu, and Val, play an important role in GfunOBP2’s binding to odorant ligands.【Conclusion】GfunOBP2 is mainly expressed in the antennae of adults of G. funebrana and the corresponding recombinant protein has strong binding affinities to 12 of the 35 volatile compounds of apple trees, and has moderate binding affinities to 10 compounds, indicating that GfunOBP2 plays an important role in the process of perceiving and recognizing the volatile compounds of host plants. This study provides a theoretical basis for confirming that Plus-C OBP was involved in the peripheral olfactory communication of G. funebrana.

Effect of Dextran Modified Phosphate Fertilizer on the Winter Wheat Yield and Fertilizer Utilization Rate

YAN YanGe, ZHANG ShuiQin, LI YanTing, ZHAO BingQiang, YUAN Liang

Scientia Agricultura Sinica. 2023, 56(12): 2317-2328. doi:10.3864/j.issn.0578-1752.2023.12.007

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【Objective】The aim of this study was to investigate the effects of dextran modified phosphate fertilizer prepared by the reaction between dextran and phosphate fertilizer with different polymerization degrees on the growth and soil phosphorus effectiveness of wheat, so as to provide the scientific support and theoretical basis for the application of dextran in phosphate fertilizer. 【Method】By using the reaction method, glucose (monomer), maltose (2-polymer), oligomaltose (≈5-polymer) and polydextrose (≈20-polymer) were added to a mixture of phosphoric acid and potassium hydroxide at 1% addition to prepare glucose-modified phosphate fertilizer (GP), maltose-modified phosphate fertilizer (MP), oligomaltose-modified phosphate fertilizer (OP) and polydextrose-modified phosphate fertilizer (PP), and the normal phosphate fertilizer (P) was prepared without the addition of dextran. The structural characteristics of the reaction between dextran and phosphate fertilizer were investigated by Fourier infrared transform spectroscopy (FTIR) and ³¹P nuclear magnetic resonance spectroscopy (³¹P NMR). Five treatments, including P, GP, MP, OP, and PP, were set up according to the principle of equal phosphorus amount, and the control (CK) was applied with only nitrogen and potassium fertilizers. The effect of different polymeric dextran modified phosphate fertilizers on wheat yield and fertilizer utilization was investigated by soil column cultivation. 【Result】(1) Compared with P, the FTIR spectra of dextran modified phosphate fertilizer showed a new vibration peak at 975 cm^-1, and the ³¹P NMR spectra showed a new displacement peak at 3.09-4.51 ppm, which might be due to the reaction between the hydroxyl group of dextran and phosphoric acid to form orthophosphate monoester. (2) Wheat yields were increased by 5.1%, 9.3%, 11.2% and 1.4% for the treatments with different polymerization degrees of dextran modified phosphate fertilizers (GP, MP, OP and PP) compared with P, respectively, mainly through the number of spikes, followed by the number of grains. (3) Compared with P, the total phosphorus uptake of wheat was significantly higher by 8.2%-21.4% under different polymerization degrees of dextran modified phosphate fertilizer treatments, among which, OP treatment was significantly higher than the other treatments. (4) The apparent phosphate fertilizer utilization rate of dextran modified phosphate fertilizer treatment was increased by 4.4-11.5 percentage points compared with P. The phosphate fertilizer bias productivity and phosphorus fertilizer agronomic efficiency were increased by 1.4%-11.2% and 1.6%-13.1%, respectively. The phosphate fertilizer utilization rate of both MP and OP treatments were significantly higher than P. (5) Compared with P, the soil fast-acting phosphorus content of dextran modified phosphate fertilizer treatment was significantly higher 10.2%-29.9%, and the OP treatment was significantly higher than the other dextran modified phosphorus fertilizer treatments. 【Conclusion】 Compared with common phosphate fertilizer, all dextran modified phosphate fertilizers with different polymerization degrees could improve wheat yield, promote the uptake and utilization of phosphorus in wheat, increase soil fast-acting phosphorus content, and reduce phosphorus fertilizer fixation. With the increase of dextran polymerization degree, wheat yield and apparent phosphorus fertilizer utilization increased first and then decreased. The best effect of dextran polymerization on the modification and efficiency of phosphate fertilizer was achieved when the polymerization degree of dextran was 4-6.

Effect of No-Tillage Combined with Mulching on the Structure and Organic Carbon Content of Aggregates in Heilu Soil of the Weibei Dry Plateau

ZHOU MingXing, DAI ZiJun, FAN Jun, FU Wei, HAO MingDe

Scientia Agricultura Sinica. 2023, 56(12): 2329-2340. doi:10.3864/j.issn.0578-1752.2023.12.008

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【Objective】The aim of this study was to clarify the effects of long-term no-tillage and mulching measures on soil aggregate structure and organic carbon content in Weibei dry plateau farmland, and to explore suitable tillage measures to improve the local soil.【Method】On the basis of continuous 16-year field positioning experiments, a total of 5 field management measures were selected as experiment treatments, including traditional tillage (CT), no-tillage and no mulch (NT), no-tillage + straw mulch (NS), no-tillage + plastic film mulching (NP), and no-tillage + straw + plastic film mulching (NSP). The 0-40 cm ring knife and profile soil samples were collected during the spring maize harvest in October 2019, and the bulk density, aggregate particle size distribution and organic carbon content were determined.【Result】(1) No-tillage and mulching measures (NT, NP, NS and NSP) affected the bulk density and aggregate size distribution of Heilu soil. No-tillage and mulching measures both increased the soil bulk density of the plough layer (0-20 cm), of which 0-10 cm increased significantly (6.8%-17.8%). The changes of bulk density and porosity of the plough layer were opposite to those of the plough layer. The proportion of micro-aggregates was significantly reduced, which promoted the transformation of micro-aggregates into macro-aggregates. The weight percentage of aggregates of each particle size in the plough layer (0-20 cm) was distributed as follows: larger aggregates (0.25-2 mm)>large aggregates (>2 mm)>micro aggregates (0.053-0.25 mm)>powder. The clay fraction (<0.053 mm), the larger aggregates and silty clay fractions in the plow bottom layer (20-40 cm) were significantly higher than the macroaggregates and microaggregates. (2) Under no-tillage and mulching measures, the organic carbon content increased with the increase of aggregate particle size. In the 0-40 cm soil layer, the organic carbon content of the aggregates of each particle size under NT treatment was significantly lower than that under CT treatment, while the NS and NSP treatments were significantly higher than those under CT treatment. (3) The particle size distribution of aggregates was the dominant factor causing the change of the nutrient contribution rate of aggregates. The total organic carbon accumulation in the plough layer was dominated by aggregates>0.25 mm, and the plough layer was mainly composed of silty clay components and organic carbon in larger aggregates. 【Conclusion】Long-term no-tillage and mulching measures promoted the transformation of micro-aggregates into macro-aggregates in the plough layer. Compared with traditional tillage, no-tillage and plastic film mulching decreased the organic carbon content of aggregates of various particle sizes in Heilong soil and in the plough layer, respectively. However, no-tillage mulching (NS, NP and NSP) increased the organic carbon content of aggregates of each particle size compared with no mulching. Straw mulching alone had the best effect, and significantly improved the bulk density and the organic carbon content of each particle size aggregate increased the most, which was the best treatment in this study.

Effects of Straw Interlayer Combined with Spring Irrigation on Saline- Alkali Soil Respiration and Its Temperature Sensitivity in Hetao Irrigation District

YU Ru, SONG JiaShen, ZHANG HongYuan, CHANG FangDi, WANG YongQing, WANG XiQuan, WANG Jing, WANG WeiNi, LI YuYi

Scientia Agricultura Sinica. 2023, 56(12): 2341-2353. doi:10.3864/j.issn.0578-1752.2023.12.009

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【Objective】This study was conducted to explore the responses of saline-alkali soil respiration to straw interlayer and irrigation regime, and to clarify the relationships between soil respiration rate and soil temperature/water content in Hetao Irrigation District. 【Method】A randomized block design was set up in 2015 with three treatments: autumn and spring irrigation (I_SA), spring irrigation without autumn irrigation (I_S), and spring irrigation plus straw interlayer without autumn irrigation (SI_S). Soil respiration, soil temperature and moisture content were measured in 2017 and 2018, and the temperature sensitivity of soil respiration rate was also estimated. 【Result】(1) Soil temperature and soil moisture content in the 0-20 cm soil layer fluctuated obviously, while it was relatively stable in the 20-40 cm soil layer. SI_S increased the soil temperature in 0-40 cm soil depth and moisture content in 20-40 cm soil depth. (2) The soil respiration rate in 2017 was higher than that in 2018. It was the highest in the flowering stage, followed by budding stage, before spring irrigation, before sowing, and harvest stage. (3) The soil respiration rates of I_S and SI_S were significantly higher than I_SA at the flowering stage (P<0.05). Compared with I_SA, I_S and SI_S increased soil respiration rate by 0.12-0.44 and 0.06-0.42 μmol·m^-2·s^-1, respectively. Compared with the I_S, the soil respiration rate of SI_S decreased by 0.01-0.49 μmol·m^-2·s^-1. These results indicated that the soil respiration rate was increased without autumn irrigation, while it was decreased with straw interlayer. (4) The soil respiration rate positively correlated with soil temperature (P<0.01), while it had no significant correlation with soil moisture content. Soil temperature at the 0-20 and 20-40 cm soil layer explained 40.74%-53.84% and 39.27%-53.46% variation of soil respiration rate, respectively. (5) The temperature sensitivity of soil respiration (Q₁₀) varied within 1.68-1.98 for different treatments, and the Q₁₀ of the 20-40 cm soil depth was higher than that of the 0-20 cm soil depth. Compared with I_SA, I_S and SI_S reduced Q₁₀. However, SI_S increased Q₁₀ compared with I_S. 【Conclusion】Overall, the straw interlayer increased the soil temperature in 0-40 cm soil depth and moisture content in 20-40 cm soil depth, reduced soil respiration rate, and increased the temperature sensitivity of soil respiration, which was used as an effective practice for water saving and emission reduction in the Hetao Irrigation District.

Optimization of Water and Fertilizer Management of Substrate Cultivated Peppers Based on Quality, Yield, and Water and Fertilizer Use Efficiency

ZHOU DaoMing, SUN Tao, ZHAO YuHong, JIA YuanJie, YANG MingFei, QU Feng, HU XiaoHui

Scientia Agricultura Sinica. 2023, 56(12): 2354-2366. doi:10.3864/j.issn.0578-1752.2023.12.010

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【Objective】The aim of this study was to optimize the water and fertilizer management of peppers, and to investigate the effects of the coupling of irrigation frequency and nutrient solution supply on the quality, yield, water use efficiency and fertilizer partial productivity of peppers grown in substrate bags. 【Method】 Kailai (37-83) RZ F1 pepper was chosen as the material in the study, the irrigation amount (IA) required to maintain the water content of the substrate at 55%-60% was set as the total daily IA of single plant, and three irrigation frequencies (IF) of single plant were to supply IA according to 1 time (IF1), 2 times (IF2) and 4 times (IF3), respectively, and two nutrient solution supply amounts (NS) were the standard Yamazaki pepper nutrient solution (NS1, i.e. 250 mL/plant per day and 500 mL/plant per day during the flowering to triple layer harvesting period and after the triple layer harvesting, respectively) and the increasing nutrient solution (NS2, i.e. the initial nutrient solution supply was 250 mL/plant per day, after each layer of pepper was harvested, the nutrient supply of single plant was increased by 50 mL, and did not increase until it increased to 500 mL/plant), for a total of six coupled treatments. The principal component analysis-technique for order preference by similarity to an ideal solution (PCA-TOPSIS), membership function analysis and grey relational degree analysis were used to comprehensively evaluate fruit quality, yield, water use efficiency and fertilizer partial productivity. 【Result】The IF had a significant effect on all quality indicators except shoulder length (P<0.01); the NS had a significant effect on vitamin C, soluble protein, capsaicin and dihydrocapsaicin (P<0.01), but had no significant effect on other quality indicators; the coupling of IF and NS showed highly significant effect on the quality indicators, except the thickness of the peel (P<0.01). At the same time, IF, NS and their coupling showed extremely significant effects on pepper yield and water use efficiency (P<0.01). The evaluation results were consistent by PCA-TOPSIS, fuzzy membership function and grey relational degree, and the top two were IF1NS1 and IF2NS2. IF1NS1 treatment had the best fruit quality of pepper, for yield, water use efficiency, and N, P, K fertilizer partial productivity were the highest, with the value of 74 482.24 kg∙hm^-2, 34.21 kg∙m^-3, 625.95 kg∙kg^-1, 679.54 kg∙kg^-1, and 367.23 kg∙kg^-1, respectively. Therefore, IF1NS1 was the optimal water-fertilizer coupling treatment. 【Conclusion】 The optimal IF and NS management of peppers grown in substrate bags were as follows: IF of single plant was to maintain the water content of the substrate at 55%-60% required IA was supplied according to 1 time, the standard Yamazaki formula nutrient solution of 250 mL/plant per day and 500 mL/plant per day was supplied from the flowering to triple layer harvesting period and after the triple layer harvesting, respectively.

Genotype Identification and Combination Analysis of Loci Related to the Peach Flesh Texture Trait via 189 Peach Accessions

WANG ZhaoHui, LI Yong, CAO Ke, ZHU GengRui, FANG WeiChao, CHEN ChangWen, WANG XinWei, WU JinLong, WANG LiRong

Scientia Agricultura Sinica. 2023, 56(12): 2367-2379. doi:10.3864/j.issn.0578-1752.2023.12.011

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【Objective】 The molecular markers and bioinformatics were used to identify the genotypes and combinations of the F-M locus, transposon insertion in YUC11 promoter region, and 9 bp insert in NAC72 coding region by 189 peach germplasms, in order to provide the theoretical foundation for the mechanism of peach flesh formation and the selection of breeding parents.【Method】PCR amplification, KASP, and HRM were used to identify genotypes. Results of loci associated with the formation of flesh trait were further validated using bioinformatics by resequencing data in 189 peach germplasms.【Result】Two genes encoding endopolygalacturonase in F-M locus, designated PGM and PGF, are associated with peach texture. Through primer amplification, 159 (84%) PGM and 99 (52%) PGF were detected in 189 peach germplasms. Four haplotypes (H0, H1, H2, and H3) were found in F-M locus, while H0 and H1 were the major genotypes. The haplotype combination of non-melting flesh, melting flesh, free-stone melting flesh were H3H3 and H2H3, H0H0 and H0H1, H1H1 and H0H1, respectively. In addition, it was found that 18 germplasms contain homozygous transposon insertion. HRM results demonstrated that 45 germplasms were homozygous insertion (early ripening), 71 germplasms were heterozygous insertion (middle ripening), and 73 germplasms were no insertion (late ripening). But six germplasm genotypes were inconsistent with the phenotypes. The most frequent genotypic combinations at the three loci of non-melting flesh, melting flesh, and free-stone melting flesh were mmffHdHdI, MMffHdHdI, MMFFHdHdL, respectively.【Conclusion】The existence of four haplotypes at the F-M locus further was confirmed, 18 germplasms containing stony hard genotype were identified, a molecular marker for the identification of maturity date was developed, and genotype combinations of different germplasms at flesh-related loci were identified.

The Optimal Fermentation Technique of Radix puerariae Residues by Aspergillus niger for Dietary Fiber Modification and the Consequent Changes of Physicochemical and Functional Properties of Dietary Fibers

FU HuiZhen, DENG Mei, ZHANG MingWei, JIA XuChao, DONG LiHong, HUANG Fei, MA Qin, ZHAO Dong, ZHANG RuiFen

Scientia Agricultura Sinica. 2023, 56(12): 2380-2394. doi:10.3864/j.issn.0578-1752.2023.12.012

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【Objective】 The objectives of the present study were to screen the microbial species suitable for modification of dietary fiber (DF) of Radix puerariae residues, to establish the optimum fermentation conditions, and to clarify the changes of microstructure, physicochemical and functional properties of Radix puerariae residues DF before and after fermentation modification.【Method】Aspergillus niger, Rhizopus oryzae, Trichoderma viride and Bacillus subtilis subsp were used to ferment Radix puerariae residues, respectively, and the most effective microbial strain was screened by comparing the SDF yield of Radix puerariae residues. The single factor experiments were carried out to screen the factors influencing DF modification. Then, the Box-Benhnken central composite experiment was designed to establish the optimum DF modification conditions of Radix puerariae residues with the SDF yield as the index of evaluation. The insoluble DF (IDF) and SDF of Radix puerariae residues before and after modification under optimal fermentation conditions were extracted by enzymatic hydrolysis method. The microstructures of IDF and SDF were observed by scanning electron microscope, and the physicochemical (water holding, oil holding, and water swelling capacity) and functional properties (adsorption capacity of glucose, sodium cholate and cholesterol) of IDF samples from unfermented and fermented Radix puerariae residues were analyzed.【Result】The fermentation of Radix puerariae residues, by A. niger, R. oryzae or T. viride all increased the SDF yield, and A. niger was the most effective species. However, the fermentation with B. subtilis subsp had no significant effects on the SDF yield. Therefore, A. niger was selected as the most suitable strain for Radix puerariae residues fermentation. The optimal fermentation condition determined by response surface optimization was as follows: The ratio of solid-liquid was 1:5.8 (m/v), the inoculation volume was 4.9% (v/v), the fermentation time was 100 h, and the fermentation temperature was 24.9 ℃. Under this condition, the yield of SDF increased from 6.34% to 13.75%, while the ratio of IDF/SDF decreased from 6.14 to 2.83. Both IDF and SDF extracted from fermented Radix puerariae residues by A. niger showed more porous microstructure than those from unfermented Radix puerariae residues. Fermentation of Radix puerariae residues increased the water holding and swelling capacity of its IDF by 20 percent approximately, while it showed no significant effects on the oil holding capacity. In addition, after fermentation, the adsorption capacity of IDF of Radix puerariae residues for glucose increased by 70%, and the adsorption capacity for cholesterol increased by 44% and 28% at pH 2.0 and pH 7.0, respectively. 【Conclusion】 A. niger could modify DF of Radix puerariae residues more effectively than other 3 microbial strains. Under the fermentation condition, the SDF yield of Radix puerariae residues increased by 2.17 times after modification by A. niger, and the physicochemical and functional properties of its IDF were also improved significantly.

Effect of Heat Stress on DNA Methylation of GNAS Promoter Region in Dairy Cows

CHEN YuMei, ZHANG CongCong, HU LiRong, FANG Hao, DOU JinHuan, GUO Gang, WANG Yan, LIU QiaoXiang, WANG YaChun, XU Qing

Scientia Agricultura Sinica. 2023, 56(12): 2395-2406. doi:10.3864/j.issn.0578-1752.2023.12.013

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【Objective】Heat stress has seriously impaired the production and health of dairy cows, causing the subsequent limitation in sustainable development of dairy industry. DNA methylation is an important epigenetic regulatory mechanism involved in an animal’s heat stress response, but the potential functions and molecular mechanisms of which are not clear. The current study was conducted to detect the DNA methylation related to heat stress in dairy cows and to identify target genes related to DNA methylation, so as to provide a better insight into the epigenetics mechanism of heat stress in dairy cows.【Method】In the study, 24 Chinese Holstein lactation cows (same lactation stage and same parity) in Sanyuan dairy farm were used for the blood samples collection in heat stress period (July in the summer of 2017) and non-heat stress period (April in spring 2017), respectively, followed by DNA extraction. To explore the DNA methylation differences in dairy cows from different heat stress period, 15 of 24 animals were randomly assigned to 3 groups (N=5 animals/group), 5 DNA samples in one group were mixed together to get a single pooled DNA sample, thus 6 pooled DNA samples including 3 from spring and 3 from summer were used for the DNA methylation detection by the whole-genome bisulfite sequencing (WGBS), then the differential methylation region (DMR; 1000 bp windows, 500 bp overlap, P<0.05) and key gene were identified. PROMO and Methprimer software were used to predict transcription factor binding sites and CpG islands, respectively. Then, the bovine mammary gland epithelial cells (Mac-T) were treated at 39 ℃ for 24 h, 48 h, and 72 h, and the cell viability were detected by MTT method. Finally, using the bisulfite sequencing PCR (BSP), the methylation levels of target gene promoter in 24 dairy cows in spring and summer and Mac-T cells treated in 39 ℃ were examined, respectively. 【Result】Based on the DNA methylation analysis of WGBS data, 49 861 differential methylation regions (DMRs) associated with heat stress were identified. One of DMRs was attributed to the promoter area of GNAS complex locus (GNAS), whose methylation level significantly increased in heat-stressed animals (P<0.001). Also, there was a 352 bp CpG island in the promoter of GNAS containing potential binding sites for Sp1, C/EBP and other important transcription factors. Further the methylation status of the GNAS gene promoter region in heat stressed dairy cows were verified by BSP, and the average methylation level in all cytosine of 31 CpG sites was higher in heat stress cows than that in control groups (P<0.05), which corresponding to the above WGBS results. Moreover, the 21 (-113 bp, Chr13:57532733) and 27 (-63 bp, Chr13:57532683) CpG sites showed significant differences between the spring and summer groups (P<0.05). In Mac-T cells, after 48 h and 72 h heat treatment, the cell viability decreased significantly (P<0.01), but the overall CG methylation level of 31 CpG sites in the GNAS gene promoter region increased significantly (P<0.05), and also the similar significant methylation changes appeared in the site 21 and 27 CpG in cell. 【Conclusion】 Heat stress increased the methylation levels of the promoter region of the GNAS in dairy cows as well as in cells, which indicated that GNAS was a potential target gene regulated DNA methylation in heat stress response of dairy cows.

Effect of Valproic Acid on Reprogramming of Bactrian Camel Fibroblasts

ZHANG QiRan, LI ZongShuai, MA Tian, LI YiNa, ZHAO XingXu, ZHANG Yong

Scientia Agricultura Sinica. 2023, 56(12): 2407-2420. doi:10.3864/j.issn.0578-1752.2023.12.014

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【Objective】 To improve the efficiency of the reprogramming process of Bactrian camel fibroblasts and to reduce the risk of tumorigenesis caused by the introduction of proto-oncogenes. In this experiment, valproic acid (VPA) was added to the fibroblast reprogramming process to explore the effect of small molecules on the reprogramming of Bactrian camel fibroblasts. 【Method】 Given this, March-aged Bactrian camel fetal fibroblasts were used as test materials, combined with classic induction combination OSKM (Oct4, Sox2, Klf4, and c-Myc) and EGFP five retrovirus reprogramming of Bactrian camel fibroblasts (OSKM group), and the cells by adding VPA treatment for 7 days after the second viral infection (OSKM+VPA group) were collected. Endogenous and exogenous genes were examined by using PCR to confirm the modification effect of retrovirus on Bactrian camel fibroblasts. Eight genes were randomly selected from those more significantly affected by VPA. According to RNA-seq data, whether their trends before and after VPA addition were consistent with the trends of RNA-seq data was checked to verify the accuracy of RNA-seq data. The transcriptome sample genes were classified by GO analysis and significant enrichment pathways for target genes were clarified by using KEGG pathway enrichment analysis and hypergeometric validation analysis. Total RNA was extracted from the collected cells, and then, combined with RNA-seq and Real time-quantitative interpretation (RT-qPCR) techniques to detect the effect of VPA on the reprogramming of Bactrian camel fibroblasts. 【Result】 It was detected by using PCR that the expression of endogenous and exogenous genes in different groups. The results showed that nSox2, Sox2, Oct4, Klf4, and c-Myc genes were expressed in both OSKM and OSKM+VPA groups, and the expression in OSKM+VPA group was higher than that in the OSKM group, while they were not expressed in BCEFs group. Eight genes were randomly selected for testing, and the results showed that: three genes of TP53, CCNB1, and CCD20 were down-regulated in expression after the addition of VPA, which were related to the cell cycle signaling pathway. S100A4, CKS2, VIM, and MMP9 genes signaling were down-regulated in expression, which was related to the phenotypic characteristics of cancer; VEGFC gene expression was up-regulated in the PI3k-Akt signaling pathway. This expression trend was consistent with the trend of the histological data. The results showed that the expression of proliferation genes Mki67 and PCNA were down-regulated, while the expression of apoptosis gene CASP7 was up-regulated after the addition of VPA. KEGG and hypergeometric validation analyses of the transcriptome data were performed, and 959 differentially expressed genes were screened according to the analysis results, which were enriched in 276 signaling pathways, including eight signaling pathways with Q values less than 0.05: steroid biosynthesis, cell cycle, PPAR signaling pathway, progesterone-mediated oocyte maturation, fatty acid metabolism, ECM-receptor interactions, cell adhesion molecules, and cholesterol metabolism. The 26 differentially expressed genes related to cell cycle, fatty acid metabolism, cell adhesion molecule, and cholesterol metabolism were screened, and four of which were randomly selected for testing, showing that VPA upregulated the expression of L1CAM, CNTN1 and NFASC genes in the Bactrian camel fibroblast adhesion molecule signalling pathway and enhanced intercellular interactions. It was also upregulated that the expression of CD36 gene in the fatty acid signaling pathway. 【Conclusion】 The results showed that the VPA blocked cell before the split phase to reduce risk differentiation during the process of reprogramming. Meanwhile, VPA affected several signaling pathways in the reprogramming process of Bactrian camel fibroblasts, and regulated the expression trend of related genes in the signaling pathways, which effectively improved the reprogramming efficiency of the cells and played an important role in the reprogramming of Bactrian camel fibroblasts.

Development and Application of a Universal iELISA Antibody Assay for Abortion-Causing Salmonella in Equidae

GUO Kui, ZHANG ZeNan, LI ShuaiJie, CHU XiaoYu, WANG YaoXin, GUO Wei, HU Zhe, WANG XiaoJun

Scientia Agricultura Sinica. 2023, 56(12): 2421-2430. doi:10.3864/j.issn.0578-1752.2023.12.015

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【Objective】The aim for this study was to identify the predominant antigen of Salmonella and to develop a sensitive, specific and universal iELISA assay method for the rapid and accurate detection of Salmonella antibodies in equidae.【Method】For the purpose of screening out dominant antigens for Salmonella Abortusequi, the immunoprecipitation (pull down) tests were performed using Salmonella Abortusequi positive/negative sera with whole bacterium antigens of Salmonella Abortusequi. Then, amino acid sequence alignment of the dominant antigen were compared with Salmonella Typhimurium, Salmonella Dublin, and Salmonella Enteritidis to verify itsconservative. Three pairs of specific primers were designed and synthesized according to the nucleotide sequence of the full ompA gene published in GenBank. Three ompA genes with different lengths were amplified by PCR, and then cloned into pET28a vector and transformed Escherichia coli Rosetta (DE3) competent cell. The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by IPTG. The reactivity of the purified protein was verified using S. Abortusequi, Salmonella Typhimurium, Salmonella Dublin (S. Dublin), and S. Enteritidis serums, and one negative serum by Western blot. An indirect ELISA method for the diagnosis of equine abortion salmonellosis was developed by optimizing the amount of coating antigen, serum and secondary antibody concentrations using the purified ompA3 protein as the coating antigen, and evaluating the specificity and sensitivity of the iELISA, and finally applying the iELISA to detection of clinical samples.【Result】In this study, the ompA dominant antigen of S.Abortusequi was screened. S.Abortusequi ompA was conservative and showed 99.4%-100% identical with Salmonella Typhimurium, Salmonella Dublin and S.Enteritidis strains at the amino acid level. Three target genes were successfully obtained by PCR amplification. Three recombinant plasmids, including pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3 were successfully constructed. The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by addition of IPTG to a final concentration of 0.6 mmol·L^-1 for 5 h at 24 ℃. The recombinant ompA1 and ompA2 were obtained in an inclusion and soluble recombinant ompA3 proteins. The soluble recombinant ompA3 were identified by Western blot, which had a specific reaction with four Salmonella positive serums, therefore, the ompA3 was considered as a potential target candidate for serological detection of Salmonella. An iELISA method was developed in a maximum P/N ratio using the coating antigen at a concentration of 1 μg·mL^-1, a serum dilution of 1﹕200 and secondary antibody was 1﹕10000. The cutoff value was 0.143, and an OD₄₅₀ value over 0.143 was considered as positive. The specificity test showed that the coated antigen did not cross-react with the positive serum of common equine infectious diseases. The iELISA provided better sensitivity by detecting antibodies in intravenously infected horses, as the iELISA could continue to monitor antibody positivity up to 116 days, 47 days longer than microagglutination test (69 days). The established iELISA method was used to detect antibodies in 180 serum samples from 8 different farms. The average positive rate of iELISA antibody was 63.3%, which was 53.9% higher than that of micro agglutination test.【Conclusion】The soluble ompA3 protein was successfully expressed, and a universal indirect ELISA antibody method was established for the diagnosis of equine abortus salmonellosis. The method enables detection of antibodies to Salmonella Abortusequi in clinical samples.The method has good specificity and sensitivity and could be a promising candidate tools for use in the monitoring of the equine abortus salmonellosis epidemic.

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