Table of Content

12 November 2023, Volume 49 Issue 11

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Construction of evaluation method for tolerance to high-temperature and screening of heat-tolerant germplasm resources of bud stage in soybean

LI Jia-Jia, LONG Qun, ZHU Shang-Shang, SHAN Ya-Jing, WU Mei-Yan, LU Yun, ZHI Xian-Guan, LIAO Wei, CHEN Hao-Ran, ZHAO Zhen-Bang, MIAO Long, GAO Hui-Hui, LI Ying-Hui, WANG Xiao-Bo, QIU Li-Juan

Acta Agronomica Sinica. 2023, 49(11):  2863-2875.  doi:10.3724/SP.J.1006.2023.34025

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The frequent occurrence of extreme high temperature (HT) events causes continuous heat damage to soybean production, which seriously damages the yield components and quality traits. The seeds are sensitive to the changes of the external environment at germination stage. The rising temperature and the accompanying drought will affect the emergence of soybean seeds. The establishment of a set of scientific evaluation methods for HT tolerance at bud stage can provide a theoretical basis for the early identification of soybean, the breeding of HT tolerance germplasm, and the study of tolerance mechanism. In this study, 385 germplasm resources varieties were selected as the experimental materials, which creating a HT environment by artificial climate incubator and subjected to HT-stress for 3 d (40℃, 16 h light /8 h darkness) at bud stage of soybean. Compared with the control (25℃, 16 h light /8 h darkness), the hypocotyl length of soybean bud stage was significantly decreased 10.9% under HT stress (P < 0.05). The indices of fresh root weight, dry root weight, and root-shoot ratio increased by 13.10%, 22.20%, and 16.90%, respectively (P<0.01). The results showed that HT-stress significantly affected the surface and underground biomass distribution of bud stage in soybean. Meanwhile, the principal component analysis for the coefficient of HT-tolerance for each trait converted 11 indexes into two principal component factors. The comprehensive evaluation value (H-value) of soybean response to HT-stress was obtained by the standardized analysis of membership function, and cluster analysis was conducted for the tested varieties based on H-value. Ultimately, 385 germplasm resources were divided into 5 grades for the HT-tolerance at bud stage in soybean [namely: Grade I (tolerance), Grade II (strong tolerance), Grade III (medium), Grade IV (strong sensitive), and Grade V (sensitive type)] and four HT-resistant varieties based on the specific performance (H245, H070, H268, and H216) were initially selected combined with the actual heat resistance performance. After the stepwise regression analysis of each index, a predictive model for the comprehensive evaluation of HT tolerance (H-value) at bud stage of soybean was established: H = 0.191 + 0.017X₁ - 0.007X₂ + 0.013X₇ + 0.027X₈ - 0.009X₁₀ (R²=0.9752). Five indexes main including hypocotyl length (X₁), main root length (X₂), hypocotyl dry weight (X₇), root fresh weight (X₈), and simplified vigor index (X₁₀) were screened out as the evaluation indexes for HT tolerance at bud stage in soybean.

Application of maize 6H-60K chip in identification of maize essentially derived varieties

TIAN Hong-Li, ZHANG Ru-Yang, FAN Ya-Ming, YANG Yang, ZHANG Yun-Long, YI Hong-Mei, XING Jin-Feng, WANG Feng-Ge, ZHAO Jiu-Ran

Acta Agronomica Sinica. 2023, 49(11):  2876-2885.  doi:10.3724/SP.J.1006.2023.23066

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The identification of maize essentially derived variety has become the hot spot in the intellectual property protection of seed industry. In order to speed up the establishment of its accurate and efficient molecular identification technology, this article used multiple types of derived varieties as research materials: Jing 2416 and Jing 2416C (two inbred lines with highly similar genetic backgrounds), Jing 724 and Jing 72464 (two inbred lines with similar genetic backgrounds), as well as 893 DH lines of genetic population constructed by Jing 724 and Jing 72464. The study researched and analyzed the potential of maize 6H-60K chip including 61,214 SNPs in identification of maize essentially derived varieties. The results were as follows: (1) There were 829 SNPs differences between Jing 2416 and Jing 2416C, the GS value (genetic similarity) was 98.7%, and 56.7% of the difference loci were concentrated in the 39 Mb region of chromosome 5. (2) There were 4912 SNPs differences between Jing 724 and Jing 72464, the GS value was 90.1%, and 44.8% of the difference loci were concentrated on chromosome 3. (3) The distribution of genetic similarity values between 893 DH lines and two parents (Jing 724 and Jing 72464) was continuous. The GS value range between 893 DH lines and Jing 724 was 88.0%-97.0%, with an average of 92.6%. The GS value range between 893 DH lines and Jing 72464 was 88.3%-98.6%, with an average of 94.5%. (4) 893 DH lines were paired comparison, a total of 398,278 pairs were compared. There were specific SNP differences between all DH lines. The GS values of 893 DH lines in pairs ranged from 87.5% to 99.9%, with an average of 94.3%. Among them, the proportion of GS value ≥ 97.0% was 8.6%, and the proportion of GS value ≥ 99.0% was 1.3%. This study showed that maize 6H-60K SNP sets could accurately evaluate the genetic background of maize derived, similar or extremely similar inbred and DH lines, identify and distinguish all materials one by one, and had the potential to further lock the linkage markers of derived traits. It is suggested that the technical system for maize essentially derived variety molecular identification based on Maize6H-60K SNP sets using chip, genotyping by target sequencing (GBTS) and other platforms should be urgently established, so as to provide technical support for intellectual property protection and variety innovation of maize varieties.

Genome-wide association analysis of morphological traits of flag leaf in wheat

WANG Rui, REN Yi, CHENG Yu-Kun, WANG Wei, ZHANG Zhi-Hui, GENG Hong-Wei

Acta Agronomica Sinica. 2023, 49(11):  2886-2901.  doi:10.3724/SP.J.1006.2023.21085

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The flag leaf of wheat is the primary functional leaf for photosynthesis and contributes significantly to yield. Therefore, it is essential to investigate the genetic process of flag leaf morphology and identify the candidate genes for flag leaf morphology-related features. We combined 90K SNP gene chips and 300 wheat varieties (lines) for genome-wide association analysis of flag leaf length, width, and area under normal irrigation (NI), and drought stress (DS) conditions in five environments. The results showed that flag leaf length, width, and area exhibited significant differences between the two moisture treatments and displayed rich phenotypic variation with the coefficients of variation ranging from 0.07-0.23 in different environments (P<0.05). Moreover, genome-wide association study (GWAS) revealed that a total of 37 stable genetic loci were significantly associated with flag leaf length, width, and area. These loci were distributed on chromosomes 1D, 2A, 2B, 3A, 3D, 4A, 5A, 5B, 6A, 6B, 7A, and 7B, with individual SNP loci explaining 3.70%-9.05% of the genetic variation, including 22 stable genetic loci detected under normal irrigation and 15 stable genetic loci detected under drought stress. Eight stable genetic loci at the same time detected under both water treatments were discovered on chromosomes 2B, 3A, 5A, 6A, 7A, and 7B, while the five stable genetic loci related by several traits were simultaneously detected on chromosomes 2B, 3A, 6A, and 7A. By analyzing haplotypes at markers with stable inheritance and high contribution, it was found that the Kukri_c1406_275 (R²=9.05%) marker was significantly associated with flag leaf length, with three haplotypes of FLL-Hap1, FLL-Hap2, and FLL-Hap3, and the wsnp_bq170165A_Ta_1_1 (R²=7.88%) marker was also detected in three haplotypes, FLA-Hap1, FLA-Hap2, and FLA-Hap3. In combination with phenotypic analysis, the flag leaf length of 300 winter wheat varieties (lines) containing FLL-Hap1 (77.78% frequency of occurrence) or FLL-Hap2 (18.89%) haplotypes was significantly higher than that of FLL-Hap3 (3.33%) haplotypes. The flag leaf area was significantly higher in haplotypes containing FLA-Hap1 (48.19%) than in haplotypes containing FLA-Hap2 (30.80%) or FLA-Hap3 (21.01%) (P<0.05). Different haplotypes were distributed differently in different winter wheat varieties (lines). Haplotype FLL-Hap1 was more frequently distributed in foreign varieties (lines), while haplotypes FLL-Hap2 and FLL-Hap3 were more frequently distributed in the northern winter wheat region and the southwestern winter wheat region, respectively. Haplotypes FLA-Hap1 and FLA-Hap2 were more frequently distributed in the southwestern winter wheat region and the northern winter wheat region, respectively, while haplotypes FLA-Hap3 were no more frequently distributed in all winter wheat regions. Searching for stable genetic loci under both water treatments yielded and screening of five candidate genes associated with flag leaf morphology, which could be used as the important genes for flag leaf-related traits.

Identification of chromosome deletion in synthesized Brassica auto-allohexaploids and its application in mapping genes of pigment synthesis

QIU Jie, WANG Tai, CAI Bo-Wei, DUAN Sheng-Xing, XU Lin-Shan, CHEN Xiao-Di, WANG Jing, GE Xian-Hong, LI Zai-Yun

Acta Agronomica Sinica. 2023, 49(11):  2902-2912.  doi:10.3724/SP.J.1006.2023.24287

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Polyploidy leads to the complexity of individual genomes and an increase of gene copies, which brings challenges to the genomic and genetic analysis. The deletion of a chromosome or chromosome fragment often leads to corresponding phenotypic changes, and the genes controlling these traits are located within the deleted fragments. According to this principle, chromosome deletion was usually used to map genes in a specific chromosome or chromosome fragment. In this study, a Brassica auto-allohexaploid (B^cB^cC^cC^cC^oC^o) was synthesized by the hybridization between B. carinata (2n = 34, B^cB^cC^cC^c) and B. oleracea (C^oC^o, 2n = 18). Two young hexaploid plants were differentiated from the callus of the same immature embryo during embryo rescue on MS medium with colchicine and hormone. However, while one plant showed purple leaves and stems and light-yellow flowers as its maternal parent B. carinata, the other plant had green leaves and stems and white flowers like the paternal parent B. oleracea. Cytological observation, fluorescence in situ hybridization (FISH), and genome re-sequencing analysis showed that a large fragment of chromosome B04 was absent in green plant. Transcriptome analysis showed DFR gene in the deletion region and MYB90 on the reference genome B03 chromosome may be the key genes controlling the purple color formation on leaves and stems, while Z-ISO and CRTISO2 were two genes within deletion region determining the formation of yellow flowers in hexaploids.

Genetic analysis and molecular identification of a multiple allele mutant of ZmMs7 gene in maize

CAO Xiao-Xiong, LIU Yi-Fan, ZHOU Yu-Qiang, WANG Jing, WU Yu-Jin, WANG Hong-Wu, LI Kun, LIU Xiao-Gang, HUANG Chang-Ling, LIU Zhi-Fang, GUO Jin-Jie, HU Xiao-Jiao

Acta Agronomica Sinica. 2023, 49(11):  2913-2922.  doi:10.3724/SP.J.1006.2023.33002

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We identified a maize male sterile mutant in the natural population, designated as ms20s1. The mutant had complete male sterility and lacked pollen grains in the withered anthers. Cytological analysis showed that, compared with the wild type, the ms20s1 mutant exhibited shrinkage locule, swollen tapetum cells, and aborted microspore at S11 stage, indicating that the ms20s1 mutant had abnormal tapetal programmed cell death and complete pollen abortion. Genetic analysis revealed that the male sterility trait was controlled by a single recessive nuclear gene. To clone the target gene, we constructed the F₂ populations by crossing ms20s1 with different inbred lines and analyzed the population genotype using genotyping by target sequencing (GBTS) technology. The gene was initially mapped to the 124.95-128.47 Mb region on chromosome 7, and the interval was narrowed down to 0.68 Mb after fine mapping. Bioinformatics analysis indicated that there was one known gene ZmMs7 in this region. The ZmMs7 gene encoded a PHD-finger transcription factor that played an important role in tapetum development and pollen wall formation. Allelism test demonstrated that ms20s1 was an allelic mutant of ZmMs7 gene. Gene sequencing results showed that the ms20s1 mutant had multiple sequence variants in the exon region, which were different from the reported mutants ms7-6007 and ms7gl, confirming ms20s1 was a new allelic mutant of ZmMs7. The discovery and identification of the ms20s1 mutant provide a new material for exploring the molecular mechanism and breeding application of maize genic male sterility.

Alkaline tolerance identification method of potato seedlings and comprehensive assessment of alkaline tolerance of 86 kinds of potato germplasms

ZHAO Peng, CHEN Guang-Xia, ZHANG Yan-Ping, YANG Xiao-Hui, LIU Fang, DONG Dao-Feng

Acta Agronomica Sinica. 2023, 49(11):  2923-2934.  doi:10.3724/SP.J.1006.2023.24278

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China is facing a serious land salinization problem. The area of saline-alkaline land reaches 100 million hectares, of which about 33 million hectares of saline-alkaline land could be used. Previous researches mostly concentrate on saline tolerance in potato, only a few research focuses on alkaline tolerance. In this study, the morphological traits such as plant height, shoot fresh weight, root fresh weight, and rooting rate of five different varieties of potato tissue culture seedlings under different concentrations of NaHCO₃ simulated alkaline stress were measured, the corresponding coefficient of variation values were also calculated. Combined with coefficient of variation and the effective growth rate, the suitable simulated alkaline stress for alkaline tolerance assessment of potato tissue culture seedling was 5 mmol L^-1 NaHCO₃. Eighty-six kinds of tetraploid potato germplasms were treated with 5 mmol L^-1 NaHCO₃, alkaline tolerance coefficient of phenotypes including plant height, shoot fresh weight, root fresh weight, and rooting rate were measured. After principal component analysis and conversion by membership function, comprehensive assessment of alkaline tolerance (defined as A-value) was calculated. The A-values of Zhongshu 3 and Beifang 008 were the lowest and highest, respectively. Cluster analysis of comprehensive assessment of alkaline tolerance indicated that 86 kinds of tetraploid potato germplasms could be classified into 5 groups. Among them, the potato varieties (lines) with strong alkaline tolerance were Beifang 008, SDP632, SDP562, SDP274, SDP869, and SDP750. The regression equation between A-values and alkaline tolerance coefficients of phenotypes was established by stepwise regression analysis, A = 0.214X₁ + 0.341X₂ + 0.398X₃ - 0.177X₄ - 0.026. The four independent variables were the alkaline-tolerance coefficient of plant height, fresh weight of stem and leaf, fresh weight of root, and rooting rate. The comprehensive assessment system of alkaline tolerance of potato tissue culture seedlings established in this study could be helpful for the preliminary assessment of potato alkaline tolerance. The identified alkaline stress sensitive and tolerant gremplasms could be further used for the study of molecular regulation mechanism of alkaline stress tolerance and alkaline resistance breeding.

Mining candidate genes related to soybean regeneration based on BSA-seq method

ZHAO Yu-Jing, ZHANG Bin-Shuo, SU An-Yu, YU Zhen-Hai, LI Jia-Huan, LIN Yang, ZHANG Yan-Ting, WU Xiao-Xia, ZHAO Ying

Acta Agronomica Sinica. 2023, 49(11):  2935-2948.  doi:10.3724/SP.J.1006.2023.24276

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Transgenic breeding technology can improve soybean directionally, and provide a new idea for improving soybean yield. In order to search for the genes related to soybean regeneration, explore the rules of soybean regeneration, and improve the efficiency of genetic transformation, we conducted soybean organogenesis experiment with 200 materials including DN50 (a material with strong regeneration ability), Keburi (a material with weak regeneration ability), and RILs of its offspring with strong regeneration ability. Compared the differences in regeneration ability between different genotypes, 20 extreme materials each were screened. Preliminary localization of soybean regeneration candidate genes by BSA-seq (bulked segregant analysis sequencing) technology, 88.04 G clean data were obtained in the 2 Mb interval with an average sequencing depth of 20.03 ×. The differentially expressed genes were mainly enriched in 20 items such as cellulose microfiber tissue, plant type cell wall tissue, or biogenesis, among which there were 6 genes in plant type cell wall tissue or biogenesis item significantly enriched. The tissue expression analysis of 6 genes showed that the relative expression level was high during cluster bud elongation, which indicating that it played a role in the process of soybean regeneration and might be the key gene affecting soybean regeneration. This study provides the basic materials for breeding new regenerated soybean varieties, and confirms the feasibility of BSA-seq technology in mining regenerated genes.

Identification and relative expression pattern of PLA1 gene family in flax

ZHAO Li-Rong, LI Wen, WANG Li-Min, QI Yan-Ni, LI Wen-Juan, XIE Ya-Ping, DANG Zhao, ZHAO Wei, ZHANG Jian-Ping

Acta Agronomica Sinica. 2023, 49(11):  2949-3295.  doi:10.3724/SP.J.1006.2023.24224

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Phospholipase A1 (PLA1) plays an important role in plant growth and development and stress responses. However, there is no function study of PLA1 in flax. In this study, we identified the PLA1 gene family in seven species including flax by bioinformatics, and analyzed the sequence characteristics, phylogenetic evolution, cis-acting elements, collinearity, and replication events of PLA1 (LuPLA1) genes in flax. Transcriptome data were used to analyze the relative expression patterns in different genetic backgrounds and organs, and qRT-PCR was used to analyze the relative expression patterns in different tissues and developmental stages, and under different stress treatments. The results showed that there were 14, 21, 15, 20, 41, 18, and 35 PLA1 members identified in flax, Arabidopsis, maize, rice, soybean, castor, and cassava, respectively. LuPLA1 were distributed on 8 chromosomes. Sequence analysis showed that except for LuPLA1-7 and LuPLA1-10, all the other members had introns and most of them had one intron, with protein length of 388-1759 aa, isoelectric point of 5.99-9.19, and molecular weight of 41.55-192.61 kD. All the LuPLA1 proteins were hydrophilic, and most of them localized in vacuoles and contained 4-25 motifs. Phylogenetic analysis revealed that the PLA1 proteins were divided into four clades, and clade IV had the largest number of PLA1 members. Collinearity analysis indicated that LuPLA1 had homologous genes in Arabidopsis, maize, rice, cassava, and soybean, and had the most homologous genes with soybean and cassava. There were two tandem duplicated gene pairs and seven segment duplicated genes pairs of LuPLA1 family members, and all the duplicated genes underwent purification selection. Transcriptome analysis showed that most LuPLA1 members exhibited organ-specific expression patterns. The promoter regions of LuPLA1 contained a large number of hormone and stress response elements. The qRT-PCR further confirmed that the relative expression of LuPLA1 genes were induced by hormone, drought, high salt, low temperature, and high temperature. LuPLA1-1 was induced by IAA. LuPLA1-1 and LuPLA1-6 were induced by NAA. LuPLA1-1, LuPLA1-5, and LuPLA1-6 were induced by GA₃. LuPLA1-12/14 was induced by NaCl and PEG. Except for LuPLA1-2/4, the other genes were induced by high temperature. All LuPLA1 members were induced by low temperature, and the response to low temperature was the most obvious. This study laid a foundation for further analysis of the function of LuPLA1 gene family.

Gene expression characteristics of TaNRT/TaNPF family in wheat cultivars with different nitrogen efficiency

WANG Lu-Lu, YI Zi-Bo, WANG Hao-Zhe, NAI Fu-Rong, MA Xin-Ming, ZHANG Zhi-Yong, WANG Xiao-Chun

Acta Agronomica Sinica. 2023, 49(11):  2966-2977.  doi:10.3724/SP.J.1006.2023.21063

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Nitrogen is one of the essential elements for wheat growth and development, and NO₃^--N is the main form of nitrogen that wheat obtains from soil. NRT/NPF genes family encode membrane transporters, which are mainly involved in NO₃^--N absorption, transport, and allocation in plants. In order to understand the relationship between NRT/NPF family and nitrogen utilization in wheat, the relative expression characteristic of TaNRT/TaNPF family in flag leaves of N-efficient wheat cultivars Zhoumai 27 (ZM27) and N-inefficient wheat cultivars Aikang 58 (AK58) at flowering stage were studied with the second-generation sequencing technology. The results showed that 386 genes of TaNRT/TaNPF family were identified in the second generation transcriptome database. Compared with AK58, there were 27, 16, and 23 differentially expressed genes in ZM27 in reducing (N120), normal (N225), and excessive (N330) nitrogen treatments. There were 16 (59.26%), 12 (75%), and 19 (82.61%) up-regulated genes in ZM27, respectively. Seven genes were down-regulated in ZM27 in reducing nitrogen treatment. The relative expression level of TaNPF8.1 was the highest and significantly up-regulated by 1.5 times in nitrogen excessive condition. In conclusion, the relative expression of TaNRT/TaNPF family genes was regulated by nitrogen application rate and cultivar. Wheat network database showed that the relative expression of TaNRT/TaNPF family had tissue specificity and chromosomal preference. The highest expression level of TaNPF8.1 in flag leaf was located on chromosome 3A, and the root specific expression TaNRT2.2 and TaNRT3.1 were mainly distributed on chromosome 6. The stem specific expression of TaNPF4.5 was mainly distributed on chromosome 2. The qRT-PCR of TaNRT/TaNPF genes were consistent with the results of the second-generation transcriptome and network data. Interaction analysis of TaNPF8.1, TaNPF4.5, and TaNRT3.1 revealed that NO₃^--N transport may also require the collaborative participation of transcription factor MYB, chlorophyll A-B binding protein, and chaperone protein. These findings laid a foundation for further studies on the relationship between TaNRT/TaNPF family expression and nitrogen uptake and utilization.

Genome-wide identification of potato (Solanum tuberosum L.) PAL gene family and its expression analysis in abiotic stress and tuber anthocyanin synthesis

ZHU Jin-Yong, LIU Zhen, ZENG Yu-Ting, LI Zhi-Tao, CHEN Li-Min, LI Hong-Yang, SHI Tian-Bin, ZHANG Jun-Lian, BAI Jiang-Ping, LIU Yu-Hui

Acta Agronomica Sinica. 2023, 49(11):  2978-2990.  doi:10.3724/SP.J.1006.2023.24247

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The phenylalanine ammonia-lyase (PAL) is the rate limiting enzyme and key enzyme in phenylpropane metabolism pathway, which plays an important role in plant growth. In this study, a total of 8 gene family members (StPALs) in potato (Solanum tuberosum L.) were identified by BlastP and Hmmer 3.1 software, and their bioinformation were analyzed by the ExPASy, CELLO, PlantCARE, and other online tool. We analyzed the relative expression pattern of StPAL genes in different tissues of double monoploid (DM) potato, as well as under abiotic stresses by RNA-seq in Potato Genome Sequencing Consortium (PGSC) database. We performed RNA-seq on white, red, and purple tuber skin and flesh of three potato cultivars, and the relative expression levels of StPALs genes in different colors tubers (flesh) of three hybrid progeny potatoes were detected by qPCR. StPAL genes were distributed on chromosomes 3, 5, 9, and 10, and eight StPALs were closely related to tobacco (Nicotiana tabacum) PAL. The cis-acting elements revealed that the promoter regions of StPAL genes contained many elements, including light response, stress response to adversity, hormone response, growth and development, and transcription factor binding elements. The results showed that StPAL2 was specifically expressed in stolons, and StPAL3/5/8 were mainly expressed in tubers and stolons. The relative expression level of StPAL3 gene were down-regulated under mannitol treatment, and the relative expression levels of StPAL3 and StPAL8 genes were up-regulated under heat stress. The above results suggested that StPALs might be involved in the tuber growth and abiotic stress response. By transcriptomic and qPCR analysis, the relative expression levels of 5 StPAL genes (StPAL3/4/5/6/8) were up-regulated in the flesh of color potato, suggesting that they may participate in the biosynthesis of anthocyanins in flesh. These results provide a theoretical basis for further understanding the StPAL gene family and analyzing the function of StPALs in potato.

Identification and analysis of BnKNOX gene family in Brassica napus

CHEN Wu-Jun, LIU Jiang-Dong, JIANG Kai-Xuan, WANG You-Ping, JIANG Jin-Jin

Acta Agronomica Sinica. 2023, 49(11):  2991-3006.  doi:10.3724/SP.J.1006.2023.34027

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The plant specific KNOTTED-LIKE HOMEOBOX (KNOX) protein is a transcription factor family that plays an important role in plant growth and development, as well as responses to various stresses. KNOX proteins contain four conserved domains [a three amino acid loop extension (TALE) type homeodomain (HD), ELK domain, and two subdomains (KNOX1 and KNOX2)]. At present, the comprehensive study of BnKNOX genes in Brassica napus has not been reported yet. In this study, 36 BnKNOX family members were identified in B. napus genome via bioinformatic analyses, which were classified into three sub-families (I, II, and M-type) based on sequence alignment and phylogenetic analyses. Evolutionary analysis showed that the whole genome duplication (WGD) and segmental duplication were the main driving forces for the expansion of BnKNOX gene family. Based on the RNA-seq data of developing tissues and organs of B. napus, BnKNAT25/26/29/30 were specifically expressed in endosperm and developing seeds, while BnKNAT31/32/34 were highly expressed in mature seeds. We analyzed the cis-acting elements and expression pattern of BnKNOXs under various abiotic stresses, and identified 17 BnKNOX members that might be involved in drought and osmotic stress responses.

StvacINV1 negatively regulates drought tolerance in potato

GONG Hui-Ling, LIN Hong-Xia, REN Xiao-Li, LI Tong, WANG Chen-Xia, BAI Jiang-Ping

Acta Agronomica Sinica. 2023, 49(11):  3007-3016.  doi:10.3724/SP.J.1006.2023.34015

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Plant vacuolar acid invertase catalyses irreversible hydrolysis of sucrose into glucose and fructose, which plays a vital role in plant growth, development, and abiotic stress adaption. The vacuolar acid invertase gene StvacINV1 in potato (Solanum tuberosum L.) are involved in regulating cold-induced sweetening in tubers, however the physiological role of StvacINV1 during adaptation to drought stress conditions is not yet fully understood. To investigate the mechanism of StvacINV1 regulating drought toleration under natural drought stress (water was withheld), this experiment was conducted with potato cultivars ‘Atlantic’, ‘Russet Burbank’, and their StvacINV1-RNAi transgenic lines. The results showed that drought stress strongly reduced mRNA abundance of StvacINV1 and vacuolar acid invertase activity in the leaves of the wild-type plants and StvacINV1-RNAi transgenic lines. Compared with the wild type, the transgenic lines with high interference efficiency of StvacINV1-RNAi were less prone to slower wilting, lower water loss, lower MDA content, and higher relative water content in leaves under drought stress, which indicated that the transgenic strains with high interference efficiency of StvacINV1 had higher drought tolerance than wild type. StvaclNV1 regulated negatively drought tolerance of potao. Further analysis showed that under drought stress, stomatal aperture and stomatal conductance in highly interfered StvacINV1-RNAi transgenic lines were significantly lower than wild type, whereas water use efficiency was significantly higher, which demonstrated StvacINV1 might regulate the drought tolerance of potato plants by stomatal movement. Sucrose content in highly interfered StvacINV1-RNAi transgenic lines was significantly higher than wild type under drought stress, meanwhile the exogenous high concentration sucrose treatment can induce stomatal closure, which led us to speculate that StvacINV1 was involved in regulating stomatal closure through its catalytic substrate sucrose. Compared with wild type, StvacINV1-RNAi transgenic lines were more sensitive during ABA-induced stomatal closure. In conclusion, StvacINV1 negatively regulated the drought tolerance by stomatal closure in potato plants, and StvacINV1 may be involved in regulating stomatal closure through its catalytic substrate sucrose, and StvacINV1 was involved in ABA-induced stomatal closure. This study provides a theoretical basis for breeding potato varieties resistant to both sweetening (tubers) and drought stress.

Evaluation of the forage performance of polish wheat on the Qinghai-Tibet Plateau

ZHAO Cai-Xia, SHEN Ji-Cheng, YIN Shu-Xiang, YE Fa-Hui, YANG Miao-Si, LIU Rui-Juan, LIU De-Mei, ZHANG Huai-Gang, SHEN Yu-Hu, CHEN Wen-Jie

Acta Agronomica Sinica. 2023, 49(11):  3017-3028.  doi:10.3724/SP.J.1006.2023.31003

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The Qinghai-Tibet Plateau is a typical alpine grazing area in the world, which is cold and arid, with fragile ecological environment, low level of primary productivity of natural grasslands, single suitable grass species for artificial pastures, and low quality, which can hardly suit the nutritional needs of grazing livestock. It is urgent to screen and cultivate high-quality forage crops. The objective of this study is to measure the forage traits of 40 polish wheat (Triticum polonicum L.), the entropy weighting method was applied to determine the weights of the measurement indicators, and use the gray correlation degree for a multivariate evaluation, to identify excellent forage germplasm resources of polish wheat varieties most suitable for planting in Qinghai. The main findings were as follows: (1) The grain yield of the test material had the highest weight of 7.04%, followed by 6.98% for fresh grass yield at flowering stage, also the coefficients of variation of grain and fresh forage yield were higher at 28.57% and 22.26%, respectively. (2) The forage quality analysis showed that the crude protein content of the plant and grain were higher by 23.86% and 20.28%, respectively. The acid detergent fibers and neutral detergent fiber of the plant were lower. (3) The multivariate evaluation of multiple indicators of the polish wheat revealed that three varieties with the best multivariate score among the 40 different Polish wheat were No. 9 from Argentina, No. 18 from Ethiopia, and No. 20 from Romania, and the highest multivariate score was 0.635 for No. 20, followed by 0.617 for No. 18, and 0.607 for No. 9. The materials with higher multivariate score of the test varieties and more outstanding forage traits can be the excellent test materials for the screening and breeding high quality forage grasses with high protein content on the Qinghai-Tibet Plateau according to production needs.

Identification of a rolling leaf allelic mutant e202 and fine mapping of E202 gene in rice

ZHOU Wen-Qi, QIANG Xiao-Xia, LI Si-Yu, WANG Sen, WEI Wan-Rong

Acta Agronomica Sinica. 2023, 49(11):  3029-3041.  doi:10.3724/SP.J.1006.2023.22061

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Good leaf morphology is one of the factors for ensuring crop yield. A certain degree of curling can keep rice leaves standing and not drooping, which is beneficial to improve the light area of the population, enabling it to receive more light energy and improve the photosynthetic efficiency. In this study, the mature seeds of ZH11 in rice were mutated by EMS, and a mutant named e202 with curly and straight leaves was screened from M₂ progeny. Phenotypic identification showed that e202 exhibited a variety of defective phenotypes. Compared with the wild type Zhonghua 11 (ZH11), the length and number of adventitious roots were decreased, and the panicle development was abnormal. The morphology of floret, anther, and pollen was deformed, the fertility was significantly reduced, the chlorophyll content was significantly increased, and the stomatal density was decreased. The structure of the leaves was observed by resin sectioning. Compared with the control, the number of vesicular cells increased and the volume decreased, resulting in leaf curling. Meanwhile, the anthers were sliced and observed. Normal pollen could not be formed in e202 or only very few pollen grains were formed, resulting in reduced fertility. The candidate genes were located in the physical region of 440 kb on chromosome 10 by map-based cloning. Gene sequencing analysis within the interval revealed that one base G was missing in the 1935 position of the fourth exon of Os10g0562700 (LOC_Os10g41310) gene, which resulted in the subsequent amino acid confusion. Protein translation is terminated prematurely, and e202 is a novel allelic mutant of REL2. This study provides a theoretical basis for revealing the involvement of REL2 in leaf rolling and flower development in rice.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effects of light intensity and nitrogen fertilizer interaction on carbon and nitrogen metabolism at grain-filling stage and its relationship with yield and quality of southern soft japonica rice

CHEN Xin-Yi, ZHU Ying, MA Zhong-Tao, ZHANG Ming-Yue, WEI Hai-Yan, ZHANG Hong-Cheng, LIU Guo-Dong, HU Qun, LI Guang-Yan, XU Fang-Fu

Acta Agronomica Sinica. 2023, 49(11):  3042-3062.  doi:10.3724/SP.J.1006.2023.22054

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Southern soft japonica rice Nanjing 9108 and Yangnongxiang 28 were selected as the experimental materials, and two light intensity treatments, and four nitrogen treatments were set. Light intensity treatments [100% natural light intensity (L1) and 50% natural light intensity (L2)] and four nitrogen treatments [no nitrogen fertilizer (N1) in the middle and late growth stages, one-time nitrogen fertilizer applied at the top sixth leaf stage (N2), one-time nitrogen fertilizer applied at the top fourth leaf stage (N3), and one-time nitrogen fertilizer applied at the top second leaf stage (N4)] were conducted at grain-filling stage. The difference of carbon and nitrogen metabolism at grain-filling stage and its effects on rice yield and quality under the conditions of different light intensity and nitrogen application period as well as light-nitrogen interaction conditions were investigated. The results showed that with the decrease of light intensity at grain-filling stage the net photosynthetic rate of flag leaf decreased by 7.35%-42.36% on average, sucrose phosphate synthase (SPS), and sucrose synthase (SS) had low activity, the C/N ratio of leaves decreased by 3.98-6.49, the transportation of photosynthetic products to grains decreased, and the content of grain starch (including amylose) decreased. Meanwhile, the activities of nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthetase (GOGAT) increased, plant nitrogen concentration increased, and the accumulation of protein increased relatively, which were not conducive to the formation of yield and good quality. After the application of nitrogen fertilizer at the middle and late growth stages, the activities of key enzyme in carbon and nitrogen metabolism in leaves were significantly increased, the aging of leaves was slowed down, and the grain-filling period of rice was prolonged, which were conducive to the increase of yield. With the delay of nitrogen fertilizer application period, nitrogen metabolism became more vigorous, and the protein content in grain had a relative significant increase, resulting in the decrease of the ratio of starch to protein and the ratio of amylose to protein, and the decrease of taste value. Under the experimental condition, normal light intensity combined with nitrogen fertilizer treatment (L1-N3) at the top fourth leaf stage synergistically improved the activities of key enzymes of carbon and nitrogen metabolism in leaves, thus the photosynthetic products and nitrogen-containing compounds were transported to grains in the appropriate proportions. Ultimately, the ratio of starch to protein in grain ranged from 11.43 to 12.03, and the ratio of amylose to protein ranged from 1.34 to 1.50, the rice had low hardness, high viscosity, and balance as well as good taste, high yield, and excellent quality could be obtained simultaneously.

Effects of magnesium application rate on yield and quality in oilseed rape (Brassica napus L.)

YE Xiao-Lei, GENG Guo-Tao, XIAO Guo-Bin, LYU Wei-Sheng, REN Tao, LU Zhi-Feng, LU Jian-Wei

Acta Agronomica Sinica. 2023, 49(11):  3063-3073.  doi:10.3724/SP.J.1006.2023.34051

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Magnesium (Mg) deficiency is one of the most serious problems in the main producing area of rapeseed in China. To evaluate the effectiveness of Mg fertilizer on seed yield and quality, field experiments were conducted at Jinxian, Jiangxi province and Anren, Hunan province during the 2020/2021 and 2021/2022 cropping seasons, with five Mg application rates (0, 15, 30, 45, and 60 kg MgO hm^-2). Rapeseed yield and its components, seed Mg content, oil content, and fatty acid components were analyzed. The results showed that Mg application increased rapeseed yield by 12.0%-77.1%. Mg application rate for maximal seed yield was 21.4-45.6 kg MgO hm^-2. Mg fertilizer increased the number of pods, number of seeds per pod, and 1000-weight by 5.0%-64.7%, 1.8%-19.6%, and 7.1%-8.7%, respectively, which had no significant effect on harvest density. After the application of Mg fertilizer, seed Mg concentration increased by 5.0%-30.3%, and the oil content increased by 0.63%-5.11%, but protein content reduced by 1.45%-2.34%. Seed water and glucosinolate concentration were independent of Mg nutrition. Mg application increased oil yield and protein yield per unit area by 14.4%-83.4% and 9.8%-68.1%, respectively. The amount of Mg fertilizer corresponding to the highest oil production was 30-45 MgO hm^-2. As for fatty acid composition, Mg application increased the content of oleic acid and linolenic acid in seeds by 4.4%-16.0% and 3.8%-40.8%, respectively, but decreased the content of linoleic acid by 1.2%-10.1%, which had a non-significant effect on other fatty acid components. In conclusion, Mg application was crucial to the synergistic improvement of yield and quality by improving seed yield and its components (the number of pods per plant, the number of grains per pod, and 1000-grain weight), seed oil content, and the unsaturated fatty acid content. The amount of Mg fertilizer corresponding to the highest yield and oil production was 30-45 MgO hm^-2.

Effects of water and nitrogen application on root attenuation characteristics and yield of spring maize under shallow buried drip irrigation

ZHANG Yu-Qin, YANG Heng-Shan, ZHANG Rui-Fu, LI Cong-Feng, TI Jun-Yang, GE Xuan-Liang, YANG Jing-Hong

Acta Agronomica Sinica. 2023, 49(11):  3074-3089.  doi:10.3724/SP.J.1006.2023.33009

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In order to explore the effects of shallow drip irrigation on the yield and root decay characteristics of spring corn after silking, in the Agricultural and Animal Husbandry High Tech Demonstration Park of Horqin District, Tongliao City from 2017 to 2020, traditional border irrigation conventional nitrogen application (W: 4000 m³ hm^-2, N: 300 kg hm^-2) was used as the control (CK), and drip irrigation quotas were used as the main treatments. Three levels of traditional border irrigation conventional irrigation were set: 40% (W1: 1600 m³ hm^-2), 50% (W2: 2000 m³ hm^-2), and 60% (W3: 2400 m³ hm^-2), Using nitrogen application rate as the secondary treatment, three levels of conventional nitrogen application rate [50% (N1: 150 kg hm^-2), 70% (N2: 210 kg hm^-2), and conventional nitrogen application rate (N3: 300 kg hm^-2)] were set up to determine the changes in spring maize yield under different water and nitrogen treatments. In 2019 and 2020, the BTC-100 micro root monitoring system was used to continuously monitor root system of spring maize in the 0-100 cm soil layer at the 0-50 days stage after silking. The results showed that the yield of spring maize was significantly higher in W3N3 than CK for four consecutive years, and there was not significant difference compared with W3N2, W2N3, and W2N2 treatments. The nitrogen agronomic efficiency of W3N2 was also the highest at the same time. Compared with CK, root length density, the total root surface area, and the average root diameter of W3N3 all increased in 0-60 cm soil layer while root length density, the total root surface area, and the average root diameter were less in 80-100 cm soil layer; the decrease in root length density, the total root surface area, and the average root diameter was lower from 0 d to 50 d after silking in two years, in which the root length density of W3N3 and CK decreased by 10.29% and 8.83%, and 15.04% and 14.08% and the average root decay rate of W3N3 decreased by 5.23% and 4.43% compared with CK. Under the shallow buried drip irrigation, the root length density, the average diameter, and the total root surface area of W3N3 and W3N2 were both higher than those of other treatments, whose decrease extent of average root decay rate were lower in 0-60 cm soil layer and differences were less in 80-100 cm soil layer from 0 day to 50 days after silking; During the 0-50 day stage after silking, the difference of average root decay rate between W3N3 and W3N2 was not significant, and W3N2 decreased by 5.68% and 5.44%, 9.75% and 11.98%, 7.16% and 6.77%, respectively, compared with W3N1, W2N2, and W1N2 in two years. The variance analysis showed that both drip irrigation and nitrogen application had a significant impact on yield and nitrogen fertilizer agronomic efficiency. The drip irrigation amount had a significant impact on the root length density, the total root surface area, and the average root decay rate of the 0-60 cm soil layer at the 0-50 day stage after silking. Nitrogen application amount had a significant impact on root length density of the 0-60 cm soil layers at the 0-50 day stage after silking, the total root surface area of the 0-60 cm soil layers at the 0-30 day stage after silking, and the average root decay rate of the 0-40 cm soil layer at the 0-50 day stage after silking. In summary, the root length density, the total root surface area, and the average root diameter of the 0-60 cm soil layers of W3N3 and W3N2 under shallow burying drip irrigation were relatively higher, and the decrease was lower within the 0-50 day stage after silking, the average root decay rate was low, and the yield difference between the two was not significant, however, W3N2 had higher nitrogen agronomic efficiency, which could be recommended as a suitable water and nitrogen application mode for water-saving, high-yield, and efficient cultivation of corn for the irrigation area of the Xiliaohe plain.

Effects of sowing date and density on stem, leaf growth, and yield formation in strip intercropping soybean

GAO Chao, CHEN Ping, DU Qing, FU Zhi-Dan, LUO Kai, LIN Ping, LI Yi-Ling, LIU Shan-Shan, YONG Tai-Wen, YANG Wen-Yu

Acta Agronomica Sinica. 2023, 49(11):  3090-3099.  doi:10.3724/SP.J.1006.2023.34029

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Based on the problems of weak light environment, variety and density selection in soybean-maize strip intercropping after rapeseed harvest in Southwest China, two varieties with different of maturity periods, Nanxiadou 25 (ND25, intermediate-late maturing, multi-branched) and Qihuang 34 (QH34, early maturing, less branched) were selected to explore the effects of sowing date (2021, S1: May 17, S2: May 27, S3: June 6; 2022, S1: May 10, S2: May 25, S3: June 9) and density (D1: 81,000 plants hm^-2, D2: 101,000 plants hm^-2, D3: 140,000 plants hm^-2, D4: 171,000 plants hm^-2) on the stem and leaf growth and yield formation of soybeans in strip intercropping. The results showed that at the same density, as the sowing date was delayed, the leaf area index of the two varieties decreased gradually at the beginning seed stage and the light transmittance inside the canopy increased gradually, the distribution ratio to pods of ND25 increased gradually and the lodging rate gradually decreased, while the distribution ratio to pods of QH34 to pods gradually decreased and the lodging rate gradually increased. At the same sowing date, with the increase of density, the leaf area index of the two varieties at the beginning seed stage gradually increased, the light transmittance gradually decreased, the dry matter distribution ratio of the pods gradually decreased, and the lodging rate gradually increased. Differences in light environment and dry matter distribution had different effects on the yield of different varieties. For ND25, S3 > S2 > S1 and D1 > D2 > D3 > D4, optimum yield was obtained with the yield of 1752.89 kg hm^-2 at S3 and D1. For QH34, S1 > S2 > S3 and D3 > D2 > D1 > D4, optimum yield was obtained with the yield of 1538.64 kg hm^-2 at S1 and D3. Therefore, intermediate-late maturing varieties should be appropriately sown later, while early maturing varieties should be appropriately sown earlier. Multi-branched varieties should be appropriately thinly planted, while less-branched varieties should be appropriately densely planted. The yield of soybeans can be increased through the synergy of sowing date and density for each variety.

Effects of interaction of nitrogen level and sowing rate on yield, growth, and ecological field characteristics of winter wheat

ZHOU Qi, LI Lan-Tao, ZHANG Lu-Lu, MIAO Yu-Hong, WANG Yi-Lun

Acta Agronomica Sinica. 2023, 49(11):  3100-3109.  doi:10.3724/SP.J.1006.2023.21070

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The objective of this study is to study the effects of different nitrogen level and sowing rate on the yield, growth and development, and ecological field characteristics of winter wheat, to explore the relationships between the wheat population competitiveness and its yield based on the ecological field theory, and to find a balance between nitrogen level and sowing dates for high yield and high efficiency in winter wheat. Field experiments were established as a split-plot design of five nitrogen levels (0, 90, 180, 270, and 360 kg hm^-2) and four sowing rates (135, 180, 225, and 270 kg hm^-2) from 2020 to 2022 at Wen County, Henan Province. The grain yield, nitrogen accumulation, growth and development indexes (i.e., plant height, crown width, and tillering) were measured and calculated for the aforementioned treatments. Results showed that the optimal sowing rate were both 225 kg hm^-2 for the two growing seasons, and the correspondingly nitrogen rates were 270 kg hm^-2 and 180 kg hm^-2, respectively, which achieving the highest grain yield. Compared to the other treatments, the yield increased by 7.5% and 18.1% with the optimal nitrogen and sowing rate treatment combinations. Moreover, nitrogen accumulation increased by 57.3% for nitrogen application treatments, and the potential energy of growth was increased by 72.7%. However, the tillering increased by 34.7%, and the development level per plant decreased with the sowing rates increased, the potential energy of growth decreased by 11.4%. Plant height and crown width were also significantly influenced by nitrogen level and the sowing rate. Compared to the 135 kg hm^-2 sowing rate and without nitrogen, the scope of ecological field increased by 23.0% in the other treatments. The relationship between winter wheat population ecological field area and yield was a quadratic function. When nitrogen was applied and seeding rate was increased, the population ecological field area of winter wheat increased by 116.7% and 52.5%, respectively. The appropriate N level and sowing rate in winter wheat for improved growth, yield, and ecological field characteristics in the experimental area was 239.8 kg N hm^-2 and 228.7 kg hm^-2, respectively, which could be extended to the application in the northern of Henan.

Peanut yield-related traits and meteorological factors correlation analysis in multiple environments

JIANG Xiao, XU Jing, PAN Li-Juan, CHEN Na, WANG Tong, JIANG Xiao-Dong, YIN Xiang-Zhen, YANG Zhen, YU Shan-Lin, CHI Xiao-Yuan

Acta Agronomica Sinica. 2023, 49(11):  3110-3121.  doi:10.3724/SP.J.1006.2023.24218

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Peanut (Arachis hypogea L.) is an important cash and oil crop, and increasing peanut yield has a positive effect on the ensuring edible oil security and increasing farmers income. In this study, the correlation analysis were conducted between peanut yield-related traits of 11 varieties and the meteorological data in 77 environments from 2013 to 2016. We found that main stem height and branch length were extremely significantly correlated with the average temperature, all-growth-stage sunshine duration, the average diurnal temperature variation, and the average wind speed. Branch number was extremely significantly correlated with the all-growth-stage ≥10℃ accumulated temperature and the average diurnal temperature variation. Pod number per plant and mature pod number per plant were extremely significantly correlated with the all-growth-stage sunshine duration and precipitation. Pod yield was significantly correlated with the ≥10℃ accumulated temperature, the precipitation in the whole growth period, the average diurnal temperature variation, and the average temperature. It is important guiding significance for planning peanut planting and improving peanut yield in various regions.

RESEARCH NOTES

Map-based cloning and transcriptomic analysis of a maize miniature kernel mutant mn-Mu

DING Meng-Li, WANG Ru-Yin, SHI Dong-Sheng, LI Ying-Bo, LEI Jie, CHEN Hong-Yu, SHEN Qing-Wen, WANG Gui-Feng

Acta Agronomica Sinica. 2023, 49(11):  3122-3130.  doi:10.3724/SP.J.1006.2023.23076

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Kernel is the main storage organ, which accumulates nutritional compounds and determinates maize yield. In this study, we identified a miniature kernel mutant, mn-Mu, caused by a random mutator’s insertion. Compared with wild type, mn-Mu had smaller kernels, shrunken pericarp, and decreased significantly kernel weight. Compared with wild type, starch content of mn-Mu kernels decreased slightly, while zein content increased. Cytological observation showed that the development of the central endosperm and the basal endosperm transfer layer (BETL) was impaired in mn-Mu mutant compared with the wild type. Genetic analysis revealed that mn-Mu was a single gene-controlled recessive mutant. Map-based cloning indicated that the mutant gene was positioned in 57.83-61.91 Mb on chromosome 2. In this interval, Zm00001d003776 gene, which encoded CELL WALL INVERTASE 2 (INCW2, Miniature 1, Mn1) previously reported, was found that a 1442 bp transposon derived from Bronze 1 locus inserted into its 5th exon. Allelic test validated that mn-Mu was a new allelic mutant of mn1. Furthermore, transcriptomic analysis revealed that the loss of Mn1 affected gene expression in the processes of carbohydrate metabolism, storage material accumulation, glycosyltransferase activity, cell wall biosynthesis, and cell cycle regulation. In conclusion, a new allelic mutant of maize Mn1 (mn-Mu) was identified and transcriptomic analysis was further performed, which providing a novel maize germplasm and clues toward comprehensive understanding of molecular regulation mechanism of Mn1 on kernel development.

Effects of green manure incorporation combined with nitrogen fertilizer reduction on wheat yield and nitrogen utilization in oasis irrigated area

CHAI Jian, YU Ai-Zhong, LI Yue, WANG Yu-Long, WANG Feng, WANG Peng-Fei, LYU Han-Qiang, YANG Xue-Hui, SHANG Yong-Pan

Acta Agronomica Sinica. 2023, 49(11):  3131-3140.  doi:10.3724/SP.J.1006.2023.31017

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Aiming at the problems of yield increase constrained and low fertilizer efficiency, the effect of green manure incorporation and nitrogen application on wheat yield and nitrogen utilization were investigated and the field experiment was conducted in Hexi oasis irrigated area in 2021 and 2022. Compared with the control of traditional nitrogen application mode (G0N1), setting the following treatments include green manure incorporation at 7500 kg hm^-2 (G1), 15,000 kg hm^-2 (G2), 22,500 kg hm^-2 (G3), and 30,000 kg hm^-2 (G4), and nitrogen fertilizer reduction at 153 kg hm^-2 (N2), and 126 kg hm^-2 (N3), their combined application with each other as G1N2, G2N2, G3N2, G4N2, G1N3, G2N3, G3N3, G4N3, and the combined effects on nitrogen absorption and accumulation and grain yield of wheat were analyzed. The results showed that green manure incorporation at 30,000 kg hm^-2 combined with chemical nitrogen fertilizer reduction by 15% (G4N2) can effectively improve crop yield and promote nitrogen use efficiency at the same time. Compared with G0N1, the grain yield and N utilization efficiency (NUTE) of G4N2 were significantly increased by 8.2% and 45.4%, respectively. Compared with G2N2, G4N3, and G3N3, G4N2 increased grain yield and NUTE by 26.1%, 19.1%, 15.5% and 28.9%, 8.4%, and 24.3%, respectively. Compared with G0N1, G4N2 significantly increased harvest index, nitrogen fertilizer partial productivity (PFP_N), and nitrogen harvest index (NHI) by 36.8%, 43.8%, and 33.9%, respectively, and compared with G3N2, G2N2, G4N3, and G3N3 by 15.0%-26.3%, 9.6%-29.2%, and 5.6%-11.2%, respectively. At anthesis and maturity stages, the AGN and NNI of G4N2 were significantly increased by 22.5% and 13.9%, 38.1% and 37.4%, compared with other treatments. Compared with G0N1, G4N2 significantly increased nitrogen translocation of stems and leaves by 35.8% and 20.4%, and significantly increased nitrogen translocation rate by 26.6% and 23.3%, and significantly increased the contribution of translocated nitrogen to grain by 29.1%, 25.7% in wheat. In conclusion, green manure incorporation at 30,000 kg hm^-2 combined with chemical nitrogen fertilizer reduction by 15% can effectively coordinate the aboveground nitrogen accumulation and translocation, significantly increased nitrogen harvest index and partial nitrogen fertilizer productivity, thus increasing wheat yield.