Scientia Agricultura Sinica

Identification and Pyramiding of QTLs for Traits Associated with Pre-Harvest Sprouting Resistance in Two Wheat Cultivars Chuanmai 42 and Chuannong 16

WANG Qin,LIU ZeHou,WAN HongShen,WEI HuiTing,LONG Hai,LI Tao,DENG GuangBing,LI Jun,YANG WuYun

Scientia Agricultura Sinica. 2020, 53(17): 3421-3431. doi:10.3864/j.issn.0578-1752.2020.17.001

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【Objective】Pre-harvest sprouting (PHS) is one of the serious problems for wheat production, which significantly reduces grain yield and end-use quality, especially in rainy or high humidity regions. The objective of this study is to identify and aggregate quantitative trait loci (QTLs) for traits associated with PHS resistance, which will provide a theoretical basis for improving PHS resistance in Sichuan wheat cultivars.【Method】A recombinant inbred line (RIL) population derived from Chuanmai 42 and Chuannong 16 was used to detect QTL and assess the new germplasm resources for PHS resistance. 127 lines in RIL population were analyzed by phenotypic and genetic identification for PHS-related traits. Seed germination index (GI), seed germination rate (GR) and seed germination rate of in each spike (SGR) in two different environments were used to evaluate PHS resistance. All QTLs for PHS resistance were mapped by an available high-density single nucleotide polymorphism (SNP, 90K). The pyramiding of the resistant QTL was also analyzed according to the genotype of every line in RILs. 【Result】There were significant difference in GI, GR and SGR between two parents. PHS resistance of Chuannong 16 was superior than that of Chuanmai 42. A total of 11 QTLs for PHS were detected on chromosomes 2B, 2D, 3A, 3D, 4A, 5A, 5B and 6B. QSgr.saas-5B was significantly associated with PHS resistance in single environment and explained 29.03% phenotypic variation. QSgr.saas-2D, QSgr.saas-3A, QGi.saas-5A and QGr.saas-5A could express stably in two environments, and the alleles of enhancing PHS resistance were from Chuannong 16. The results of genotype analysis showed that the number of resistant QTL in different lines ranged from one to nine. Six excellent lines in RILs with high resistance carried seven or eight additive QTLs for PHS resistance. These additive QTLs included the minor QTLs on chromosome 4A from Chuanmai 42 and the major QTLs on chromosomes 2D and 5B from Chuannong 16. No. 104 and No. 125 in RIL population were released in China or Sichuan province because of its high yield and PHS resistance, and were named Chuanmai 104 and Chuanmai 64, respectively. Chuanmai 104 showed high yield and good resistance for stripe rust, powdery mildew and PHS in the Sichuan provincial trials and the national trails for Upper and Middle Yangtze River region in 2010 and 2012. The QTL analysis for PHS resistance revealed that Chuanmai 104 carried seven QTLs, including four QTLs on chromosomes 2B, 2D and 5B from Chuannong 16 and three QTLs on chromosomes 4A and 6B from another parent Chuanmai 42. The pyramiding of these additive QTL alleles from each parent led directly to the character of high PHS resistance in Chuanmai 104. In recent years, Chuanmai 104 was widely used to wheat improvement in Southwest China, and 18 wheat varieties (lines) have been bred. 【Conclusion】Eleven QTLs for PHS resistance, including three QTLs from Chuanmai 42 and eight QTLs from chuannong16, were detected in this study. Four to nine resistant QTLs were generally carried by the resistant lines in RIL population. Two pyramiding lines (Chuanmai 104 and Chuanmai 64) with high PHS resistance carried seven resistant QTLs.

Cloning and Function Analysis of Sesame Galactinol Synthase Gene SiGolS6 in Arabidopsis

LIU AiLi,WEI MengYuan,LI DongHua,ZHOU Rong,ZHANG XiuRong,YOU Jun

Scientia Agricultura Sinica. 2020, 53(17): 3432-3442. doi:10.3864/j.issn.0578-1752.2020.17.002

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【Objective】SiGolS6 is a member of galactinol synthase family in sesame, which may play important role in drought stress resistance. Studying the function of SiGolS6 in plant drought stress resistance, so as to provide theoretical basis and gene resources for drought resistance improvement of sesame. 【Method】The galactinol synthase gene, SiGolS6, was isolated from sesame through reverse transcription PCR (RT-PCR). The sequence was analyzed by bioinformatics tools such as InterProScan, ClustalX2 and MEGA5.2. Then the function of SiGolS6 in plant drought resistance was characterized by phenotype analysis and physiological index measurement of SiGolS6 transgenic Arabidopsis thaliana. 【Result】The total length of the CDS sequence of SiGolS6 was 921 bp, encoding a polypeptide of 306 amino acids. The molecular weight of SiGolS6 is 35.07 kD and its isoelectric point is 4.7. Sequence analysis showed that SiGolS6 protein contained conserved glycosyl transferase domain (IPR002495), belonging to the glycosyl transferase superfamily. Based on phylogenetic tree constructed with GolS proteins from sesame and other species, SiGolS6 was highly similar to the homologous genes in potato. Six independent transgenic Arabidopsis thaliana lines were identified by hygromycin screening and PCR. Three transgenic lines (OE-2, OE-3, and OE-4) with high expression levels were identified by qRT-PCR, and used for subsequent experiment. Raffinose content in those transgenic plants was higher than that in wild-type (WT) plants. Under drought stress, the wilting degree of transgenic lines was less than that of WT lines. After 28 d of drought stress and 5 d of recovery, the fresh weight of transgenic lines was significantly higher than that of WT, but there was no significant difference under normal conditions. After 5 d of recovery, 50% of transgenic plants recovered, while the survival rate of WT was less than 10%. After 21 d of drought stress, the relative electrical conductivity, ROS accumulation and MDA content of transgenic plants were significantly lower than those of WT, while the relative activities of SOD and POD were significantly higher than those of WT.【Conclusion】Overexpression of SiGolS6 in Arabidopsis thaliana could improve the drought tolerance of transgenic plant.

Analysis of F-Box Gene Family Based on Salt-Stressed Transcriptome Sequencing in Vicia faba L.

HAO ShuLin,CHEN HongWei,LIAO FangLi,LI Li,LIU ChangYan,LIU LiangJun,WAN ZhengHuang,SHA AiHua

Scientia Agricultura Sinica. 2020, 53(17): 3443-3454. doi:10.3864/j.issn.0578-1752.2020.17.003

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【Objective】The distribution structure and evolution of F-box gene family members in Vicia faba were analyzed by bioinformatics method to study the expression patterns of family members and their responses to salt stress under different treatment times. It can provide a reference for the study of the biological function and the mechanism of F-box genes.【Method】Based on the salt-stressed transcriptome sequencing (RNA-seq) data, the NR, Swiss-Prot, PFAM and NCBI websites were used at the same time to screen and annotate the F-box genes of Vicia faba. The softwares including Web Logo 3, Prot Comp 9.0, MEGA-X and MEME were also applied to analyze the bioinformatics of conserved domain, subcellular localization, phylogenetic tree and Motif. Based on salt stress transcriptome data, the differential expression patterns of F-box gene family in Vicia faba (yz17134 salt tolerance and yz17078 salt intolerance) under salt stress were analyzed, and real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the specific expression of part family members at 16 h and 24 h.【Result】Based on salt-stressed transcriptome sequencing (RNA-seq) data, 161 Vicia faba F-box genes were annotated and all contain F-box conserved domain. According to different C-terminal domains, they were divided into 11 subfamilies (FBX, FBXFBA, FBXLRR, FBXPP2, FBXKelch, FBXTUB, FBXFBD, FBXDUF, FBXACTIN, FBXWD40, and FBO). The analysis of the conserved domain showed that the F-box conserved Motif contained an extremely conserved tryptophan residue. By comparing and analyzing the evolutionary tree constructed by the F-box family of Vicia faba and the F-box family of Arabidopsis thaliana, it was found that most of the genes in the same C-terminal domain were clustered together. The results of subcellular localization prediction showed that 124 F-box genes were located outside the cell, and 37 F-box genes were located in the nucleus. The analysis of gene structure showed that there were no introns in the DNA sequences of the F-box family genes of Vicia faba, and all of them were composed of UTR zone and CDS zone. Analysis of F-box differential expression patterns based on salt-stressed transcriptome data showed that F-box gene expression in Vicia faba was diverse from each other at two different processing time points, the expression was more obvious at 16 hours after salt treatment. The results of qRT-PCR analysis showed that there were five different genes in the F-box family. The expressions of Vf056266.1, Vf062764.1 and Vf024236.1 were all up-regulated at 16 hours after salt treatment, Vf060904.1 and Vf045761.1 were both down-regulated at 16 hours after salt treatment.【Conclusion】161 Vicia faba F-box genes were identified by family annotation, and they were identified by family annotation, which were Evolutionarily divided into 11 subfamilies. 5 important genes were obtained through a series of bioinformatics analysis. What’s more, there exist difference among the expressions in diverse salt treatment time.

Isolation, Identification, and Response to Abiotic Stress of MsWRKY42 Gene from Medicago sativa L.

LIU JiaoJiao,WANG XueMin,MA Lin,CUI MiaoMiao,CAO XiaoYu,ZHAO Wei

Scientia Agricultura Sinica. 2020, 53(17): 3455-3466. doi:10.3864/j.issn.0578-1752.2020.17.004

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【Objective】The WRKY gene family in plants encodes a large group of transcription factors (TFs) that play essential roles in diverse stress responses, developmental and physiological processes. It laid a foundation for further research on the role of WRKY transcription factor in alfalfa stress-resistant molecular regulation by analyzing the role of MsWRKY42 transcription factor in alfalfa. 【Method】MsWRKY42 sequence was obtained by homologous alignment and sequence characteristics of it were analyzed by online bioinformatics tools. The phylogenetic tree of MsWRKY42 and Arabidopsis WRKY genes was constructed by MEGA-X. The putative cis-elements in promoter region of MsWRKY42 was analyzed by PlantCARE. The real-time quantitative PCR (qRT-PCR) was used to analyze the expression pattern of MsWRKY42 in different organs and its response to abiotic including NaCl (0.3 mol·L^-1), PEG(15%), 4℃, 40℃, low phosphorus and ABA (0.1×10^-3mol·L^-1). The fusion expression vector of pCAMBIA1300- WRKY-GFP was constructed and delivered into Nicotiana benthamiana by Agrobacterium mediated method to determine the subcellular localization of WRKY protein. The yeast one-hybrid technique was used to analyze the binding activity of MsWRKY42 and the cis-acting element W-box.【Result】The gene contained a 1 692 bp open reading frame encoding 563 amino acids. Multiple sequence alignment and phylogenetic tree analysis results indicated that the protein is a member of the IIb sub-group WRKY family and contains a WRKY conserved domain and a C₂H₂ zinc finger motif. It has the highest similarity to AtWRKY42 in the Arabidopsis family, therefore, named as MsWRKY42. A variety of cis-acting elements were identified in the promoter region of MsWRKY42, including regulatory elements such as stress response, hormone response, and diurnal regulation. Real-Time PCR analysis showed that MsWRKY42 had the highest expression in roots and leaves. The expression of MsWRKY42 was up-regulated by NaCl, PEG, low temperature, high temperature, low phosphorus and ABA treatments. Subcellular localization indicated that MsWRKY42 was mainly located on the nucleus in plant cells. The results of the binding activity analysis showed that MsWRKY42 was able to specifically bind to W-box.【Conclusion】MsWRKY42 is a typical transcription factor, which can specifically bind to cis-acting elements, and the protein is localized in the nucleus. The gene was expressed in different tissues of alfalfa, and was induced by NaCl, PEG, high temperature, low phosphorus and ABA treatment. It is speculated that the MsWRKY42 gene may play a role in response to multiple stress defense responses as a WRKY transcription factor.

Relationship Between Plant Dry Matter Accumulation, Translocation, Soil Water Consumption and Yield of High-Yielding Wheat Cultivars

TONG Jin,SUN Min,REN AiXia,LIN Wen,YU ShaoBo,WANG Qiang,FENG Yu,REN Jie,GAO ZhiQiang

Scientia Agricultura Sinica. 2020, 53(17): 3467-3478. doi:10.3864/j.issn.0578-1752.2020.17.005

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【Objective】By clarifying the relationship between dry matter accumulation, transportation of wheat plants and soil water change and yield formation, this paper intended to tap the production potential of cultivars, and providing the theoretical basis for improving the yield of winter wheat.【Method】Four wheat cultivars with different yield levels were selected, Yannong999, Shannong29, Hannong1412 and Liangxing67, and the field experiments were conducted in Hongtong county, Shanxi province from 2016 to 2018. The differences of dry matter accumulation and transportation, soil water consumption and their relationship with yield formation of different cultivars were compared to reveal the reasons for the differences in yield and water use efficiency among cultivars.【Result】For two consecutive years, the yield of Yannong999 and Shannong29 were higher than 9 000 kg·hm^-2, reaching a super high yield level; The yield of Hannong1412 was higher than 8 000 kg·hm^-2, reaching a high yield level; While the yield of Liangxing67 was lower than 7 500 kg·hm^-2, not reaching the high yield level. Compared with Liangxing67, the three high-yielding cultivars improved the dry matter accumulation in stages from sowing stage to jointing, jointing stage to anthesis, and anthesis to mature by 12%-57%, 5%-62% and 11%-47%, respectively, which significantly improved the dry matter transportation before anthesis and the dry matter accumulation after anthesis by 1%-85% and 11%-48%, respectively. The total water consumption during the whole growth stage was improved by 17%-29%. The water consumption of the two stages before anthesis was increased by 11%-41% and 8%-32%, respectively. Finally, the ear numbers was improved by 7%-24%, the grain number per ear was improved by 4%-13%, the weight of 1 000 grains was improved by 1%-9%, the yield was improved by 20%-37%, and the water use efficiency was improved by 2%-14%. Compared with the high-yielding cultivars Hannong1412, a super high-yielding cultivar, significantly improved matter accumulation from sowing stage to jointing stage, jointing stage to anthesis and the dry matter transportation before anthesis by 32%-33%, 41%-55% and 49%-50%, respectively; The water consumption of Yannong999 in the first two stages of anthesis was increased by 5%-7% and 3%-9%, respectively; The ear numbers of Yannong999 was improved by 8%-16%, and the grain number per ear was improved by 5%-6%; The yield of Yannong999 was improved by 10%-11%. Shannong29 improved the dry matter accumulation after anthesis, the water consumption after anthesis, weight of 1000 grains, and the yield by 13%, 6%-26%, 4%-6% and 5%-6% respectively. Correlation analysis of four wheat varieties in the two experimental years showed that the water consumption in the first two stages before anthesis was significantly related with the dry matter transportation before anthesis. The dry matter transportation before anthesis was significantly related to the ear numbers and yield, the water consumption after anthesis was significantly related to the dry matter accumulation after anthesis, and the dry matter accumulation after anthesis was significantly related to the weight of 1000 grains and yield. In addition, compared with Liangxing67, for the three high-yielding cultivars, every increase of 1 mm of water consumption in growth period could increase 16-40 kg·hm^-2 of wheat yield. The contribution of soil water consumption of super high-yielding cultivars to grain yield was greater, and its water use efficiency was 6%-22% higher than that of high-yielding cultivars.【Conclusion】The three high-yielding cultivars improved the dry matter translocation and water consumption in the two stages before anthesis, which was beneficial to optimize the yield and its components, so as to achieve the increase of yield and water use efficiency. There are some differences in wheat cultivars for getting high-yielding. Yannong999 had a strong ability of using soil water in early growth, which promoted the translocation of dry matter to grains before anthesis, and achieved super high-yielding by increasing the ear numbers and the grain number per ear. Shannong29 had a strong ability of using soil water in later growth stage, which promoted the dry matter accumulation after anthesis, and achieved super high-yielding by increasing the weight of 1000 grains.

Root Morphological, Physiological Traits and Yield of Maize Under Waterlogging and Low Light Stress

WANG Qun,ZHAO XiangYang,LIU DongYao,YAN ZhenHua,LI HongPing,DONG PengFei,LI Chaohai

Scientia Agricultura Sinica. 2020, 53(17): 3479-3495. doi:10.3864/j.issn.0578-1752.2020.17.006

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【Objective】With the global climate change, meteorological disasters occur frequently during the maize growth season. In order to explore characteristics of root response to rainy and low light stress, this study was carried out to investigate root morphology, physiological traits and yield of maize under waterlogging and low light combined stress at flowering stage.【Method】Taking two different maize varieties (Zhengdan958 and Yuyu22) as research objects, the experiment, including four treatments with contrast (CK), waterlogging (W), low light (S), and waterlogging and low light (WS) was conducted to compare dynamic changes of root dry weight, root length, root surface area, root volume, nodal root layer and to analyze the morphological, anatomical and physiological characteristics and yield under different stress.【Result】The root dry weight, root length, root surface area and root volume of maize were significantly decreased under combined stress, and these indicators were the largest reductions under waterlogging and low light stress, the second reductions under waterlogging stress and the smallest reductions under low light stress. Compared to CK, root dry weight were decreased by 15.21%, 5.08%, 21.07% , and root length were decreased by 14.86%, 5.52%, 18.14% in average under waterlogging stress, low light stress and combined stress, respectively. Root superficial area and root volume were decreased by 9.83%, 4.62%, 12.72% and 12.62%, 6.61%, 16.23% under three different stress (W, S, and WS). Compared to W and S stress, root dry weight, root length, root surface area and root volume were decreased by 6.64%, 3.84%, 3.21%, 4.12% and 16.55%, 13.10%, 8.41%, 10.32% under WS stress, and it was significantly different between WS and S. But there was no obviously different between WS and W. Root aerenchyma numbers, root aerenchyma areas, root porosity and crown root layers were increased significantly under waterlogging stress as well as waterlogging and low light combined stress. Compared with the contrast, root aerenchyma numbers were increased significantly by 5.29 and 10.03 times with W and WS treatment in average, respectively. Then aerenchyma areas and root porosities were increased by 5.76, 13.27 times and 8.01, 10.00 times under W and WS treatment, respectively. Crown root layers had more 1 to 2 layers under W and WS than that under CK, however, there was not different obviously between S and CK. Root physiological traits and yield of maize were decreasing significantly under waterlogging and low light stress. Especially there was the largest decreasing under combined stress. Compared with CK, root activities, total root absorbing area, root active absorbing area and ratio of active root absorption of maize were decreased significantly by 52.82%, 28.48%, 36.72% and 20.00% in average under combined stress, respectively. The change order of treatments was WS

A Method for the Automatic Determination of Scale Parameter During Segmenting Agricultural Drone Images

SHI YaJiao,CHEN PengFei

Scientia Agricultura Sinica. 2020, 53(17): 3496-3508. doi:10.3864/j.issn.0578-1752.2020.17.007

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【Objective】The automatic extraction of crop planting area in the image is of great significance for promoting the application of high-resolution drone images in precision agriculture. At present, the method based on designing segmentation evaluation function is most commonly used in the study of automatically determining segmentation scale parameters. In order to meet the needs of high-precision automatic segmentation of agricultural drone images, an improved evaluation function was proposed to solve the defects of the Weighted Local Variance (WLV) method in this study, and the proposed method was verified based on field experiments of different crops.【Method】With considering that WLV method does not consider the internal homogeneity of segmented objects, this study designed the Improved Weighted Local Variance (IWLV) method with adding the calculation of inter-object homogeneity on the basis of WLV formula. The nitrogen experiments of corn and water and nitrogen coupling experiment of wheat were designed. During corn and wheat growing season, drone images were obtained in different growth stages with crop in different vigor. Based on the obtained UAV images, different scenarios were set. The mainstream segmentation algorithm was combined with WLV method and IWLV method to perform image segmentation, respectively. Their segmentation results were compared with human-machine interactive segmentation results, and evaluated based on Single-scale Object Accuracy (SOA).【Result】The selected scale parameter by the WLV method tended too large, which resulted in under-segmentation during segmenting images. While, based on the selected scale parameter by IWLV method, the segmentation result was correspond well with human-machine interactive segmentation results. The IWLV method had higher SOA values for all designed scenarios.【Conclusion】Compared with the WLV method, the proposed IWLV method in this study had higher accuracy when determining the segmentation scale parameter.

UAV Spray Technology for the Citrus Orchard: Taking Control of the Diaphorina citri and Phyllocnistis citrella as Examples

WANG KeJian,LI Li,LÜ Qiang,YI ShiLai,ZHENG YongQiang,XIE RangJin,MA YanYan,HE ShaoLan,DENG Lie

Scientia Agricultura Sinica. 2020, 53(17): 3509-3517. doi:10.3864/j.issn.0578-1752.2020.17.008

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【Objective】The objective of this study is to explore the distribution of droplet deposition in citrus canopy and operation parameters of plant protection by the unmanned aerial vehicle (UAV) sprayer, carry out the test of control effect against citrus psyllid (Diaphorina citri) and citrus leafminer (Phyllocnistis citrella) by UAV in the orchard, evaluate the control effect, operation efficiency and comprehensive benefits of UAV spraying technology, and to provide a basis for the establishment and application of low-altitude and low-capacity UAV spray technology in citrus producing areas.【Method】The experiment was conducted at the high-yielding period of a Citrus paradisi orchard in Wan’an County, Jiangxi Province. Four rows of about 100 citrus trees of natural round shape canopy were cut into open center shape, and four rows of natural round shape were selected as control. The grid frame was set up in the canopy of the sampling plant. The grid frame was divided into three layers in vertical direction, 3×5 sampling points in each layer, a total of 45 pieces of double 4 cm × 6 cm paper cards as droplet carrier were placed on the top, middle and lower parts of citrus tree with 0.5% Temptation Red water solution as tracer. The distribution of the spray droplets in the canopy under different flight operation speeds (v₁=0.7 m·s^-1, v₂=1.2 m·s^-1, v₃=1.7 m·s^-1) and different operating heights (h₁=1.0 m, h₂=1.5 m, h₃=2.0 m) was analyzed with the six-rotor crop protection unmanned aircraft. After each treatment, the paper card was collected and scanned by 300 dpi resolution scanner to calculate the percentage of spreading area of the Temptation Red water solution on the paper card, which was calculated as the droplet coverage rate on the citrus leaves. The deposition distribution of sprayed droplets in plant canopy was analyzed and the operation parameters were selected. Through the selected operation parameters, the control test against D. citri and P. citrella was carried out in the orchard from the spring shoot germination to autumn shoot mature period. The work quantity, time and labor consuming, dosage, water consumption, pesticide price and other expenses of the plant protection spraying operations by the UAV and artificial spraying were recorded in each time. After spraying, the insect number was investigated every 15 d.【Result】Taking into consideration of the operational efficiency and effective droplet deposition, UAV spraying under the fly height of 1.0 m and the flying speed of 1.7 m·s^-1 with open center tree-shape in the citrus orchards could get the better droplet penetration and distribution. The average droplet coverage rate reached 19.1%. Using these parameters, the UAV control test against D. citri and P. citrella was carried out in citrus orchard. Compared with manual spraying operation, there was no significant difference in control effect. However, the work efficiency of UAV spraying was 45 times of manual spraying, the total work cost of UAV spraying was 63.3% of manual spraying, and the amount of pesticide consumption was only about 10% of the manual spraying.【Conclusion】Based on the suitable spray parameters and tree structure, the control effect of UAV spray technology against D. citri and P. citrella can be obtained, and UAV spray technology is good for pesticide reduction, labor-saving and efficient cultivation management in the citrus orchard.

Resistance Level, Mechanism of Alopecurus myosuroides and Control Efficacy in Wheat Field in Shandong Province

GAO XingXiang,LI Jian,ZHANG YueLi,LI Mei,FANG Feng

Scientia Agricultura Sinica. 2020, 53(17): 3518-3526. doi:10.3864/j.issn.0578-1752.2020.17.009

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【Background】As a new kind of malignant gramineous in the mainland of China in recent years, Alopecurus myosuroides has been widely distributed in Shandong, Henan, Hebei and Anhui provinces, and its distribution area is constantly expanding.【Objective】The objective of this study is to determine the resistance level, mechanism of A. myosuroides to herbicides and the control efficacy in wheat field, and to provide a theoretical basis for A. myosuroides control in winter wheat field.【Method】Whole-plant dose response experiments were conducted to determine the resistance level of nine populations to ALS inhibitor such as pyroxsulam, mesosulfuron-methyl and ACC inhibitor such as pinoxaden, clodinafop-propargyl and fenoxaprop-P-ethyl in the greenhouse, and the target gene of resistant population was detected. At the same time, herbicide control experiments were carried out before and after winter in the wheat field.【Result】The results of greenhouse experiment showed that only one of the nine populations, population 8 (JN2), had obvious resistance to the herbicides of ALS inhibitor (pyroxsulam and mesosulfuron-methyl), and the resistance index (RI) reached 47.32 and 15.97, respectively. ALS gene sequence analysis showed that Pro (CCC) to Thr (ACC) mutation at position 197 of the ALS gene occurred in the resistant population. Meanwhile, all the populations had no resistance to the ACC inhibitor pinoxaden, clodinafop-propargyl and fenoxaprop-P-ethyl. The results of field trials showed that the effect of application before winter was better than that after winter, the fresh weight control efficacy of pinoxaden before winter was 98.6% and that after winter was 89.1%. The plant control efficacy and fresh weight control efficacy of pyroxsulam and mesosulfuron-methyl before winter were 72.2%-89.3% and those after winter were 68.6%-83.2%, but pinoxaden, clodinafop- propargyl and fenoxaprop-P-ethyl all showed good control efficacy on A. myosuroides, the plant control efficacy and fresh weight control efficacy were above 96.2% before winter and 82.6%-92.2% after winter.【Conclusion】Among the nine tested populations, one population was found to have high resistance to mesosulfuron-methyl and pyroxsulam, but no resistant population was found to ACC inhibitor herbicides, such as pinoxaden, clodinafop-propargyl and fenoxaprop-P-ethyl. The results of laboratory test and field test were consistent.

Differences of Main Nutrient Concentration in Wheat Grain Between Typical Locations of the Loess Plateau

WANG Li,WANG ZhaoHui,GUO ZiKang,TAO ZhenKui,ZHENG MingJun,HUANG Ning,GAO ZhiYuan,ZHANG XinXin,HUANG TingMiao

Scientia Agricultura Sinica. 2020, 53(17): 3527-3540. doi:10.3864/j.issn.0578-1752.2020.17.010

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【Objective】This study was performed to understand the differences of wheat grain nutrient concentrations and their relations to soil nutrient and crop nutrient uptake and utilization among different locations at the same region, in order to guide reasonable fertilizer application and improve soil fertility for local farmers. 【Method】Field experiments were conducted at Yongshou and Yangling in Shaanxi province from 2017 to 2018. At each site, twenty wheat cultivars from different wheat production areas were planted under conditions of 180 kg N·hm^-2, 100 kg P₂O₅·hm^-2 and 75 kg K₂O·hm^-2. The aboveground wheat plant and soil samples were collected at maturity to determine the grain yield and nutrient concentration in different organs and soil available macro- and micronutrients, for investigating the relationships between soil nutrient supply and grain nutrient concentration at two locations. 【Result】Compared to Yangling, the grain nitrogen (N) and potassium (K) concentrations were decreased by 10.6% and 6.7% at Yongshou, respectively, but no difference was observed for phosphorus (P) concentration between two locations. Soil N and P supply capacity, N, P and K uptake and harvest index at Yongshou were higher than that of Yangling, but the total rainfall and its distribution at Yongshou were more beneficial to grain yield formation to Yangling. The increase magnitude of grain yield caused by the rainfall was larger than the magnitude of grain N and K uptake increase, and close to that of grain P uptake. Thus, the decrease of grain N, P and K concentrations was mainly attributed to the yield dilution. The concentrations of grain calcium (Ca) and magnesium (Mg) at Yongshou were 19.0% and 10.3% higher than that at Yangling, respectively, and the difference for sulfur (S) concentration was not significantly different between two locations. Soil exchangeable Mg at Yongshou was lower than that at Yangling, and no difference of soil exchangeable Ca was found between two locations. However, the lower soil pH and available K, and higher available S promoted the uptake and translocation of Ca and Mg to grain at Yongshou. Compared with Yangling, the increase of Ca and Mg absorption in wheat grains was greater than that of yield increase, and the increase of S absorption was close to that of the yield. Therefore, Ca and Mg concentration in grains increased, and the S concentration did not change significantly. The concentrations of grain iron (Fe), manganese (Mn) and copper (Cu) at Yongshou were 9.3%, 22.2% and 12.7% higher than those at Yangling, respectively, and grain zinc (Zn) concentration was 63.1% lower than Yangling. No significant difference was observed for soil available Mn between two locations, but soil available Cu and Zn at Yongshou were lower than that of Yangling. The longer filling period promoted the uptake of micronutrients in wheat grain, whereas the higher soil available P inhibited Zn uptake, this resulted in a higher Fe, Mn and Cu uptake and translocation to grain, and lower Zn uptake and translocation to grain. The higher Fe, Mn and Cu uptake in grain increased their concentrations, while the Zn concentration decreased. 【Conclusion】Therefore, the discordance between variation of grain yield and its nutrient uptake caused by different precipitation and soil nutrient supply capacities between locations were the key reason for their nutrient concentration variation in dryland. Compared with Yangling, the yield dilution influence was the main reason for the lower N concentration in wheat grain of Yongshou. It was the higher soil available P and available S supply, so that the higher P and S absorption of grain and aboveground at Yongshou did not decrease its grain P and S concentrations. The lower soil available K and Zn as well as the P and Zn antagonistic inhibited the accumulation of wheat grain K and Zn at Yongshou. Low pH and low available K promoted the plant absorption and transfer of Ca and Mg to grain at Yongshou, and the longer grain-filling period benefitted the absorption of Fe, Mn and Cu and the transfer to grain. In practical crop production, the optimized fertilization practice should be taken according to the specific soil nutrient supply and climate conditions for the purpose to coordinate the crop nutrient uptake and yield change, to produce wheat with high yield and high grain nutrient quality.

Effects of Yield and Fertilization on Yield Increase of Winter Wheat in Yangtze Valley Under Optimized Fertilization

HUANG XiaoMeng,LIU XiaoYan,CHUAN LiMin,YANG LanFang,HE Ping,WANG XiuBin,QIU ShaoJun,ZHAO ShiCheng,XU XinPeng

Scientia Agricultura Sinica. 2020, 53(17): 3541-3552. doi:10.3864/j.issn.0578-1752.2020.17.011

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【Objective】Aiming to the low nutrient use efficiency caused by unreasonable fertilization of winter wheat in the Yangtze Valley, this study summarized the yield distribution characteristics of each province in the Yangtze Valley and the stimulation effect of applying nitrogen, phosphorus and potassium fertilizer, so as to provide a scientific basis for optimizing the application fertilizer of winter wheat in the Yangtze Valley and improving nutrient management strategies. 【Method】The data of field experiments in this paper were derived from field experiments conducted by the International Institute of Plant Nutrition (IPNI) in China’s Yangtze Valley from 2000 to 2018, as well as published papers on wheat field trials from 2000 to 2018 obtained by searching key words (winter wheat, winter wheat + yield, winter wheat yield + fertilizer utilization, etc.) in CNKI database, totally 1 732 field trials. The experimental treatment included optimized fertilization treatment, farmers practices, and none nitrogen, none phosphorus and none potassium treatments based on these two treatments, which was used to explore the characteristics of attainable yield, yield response, relative yield, agronomic efficiency and partial factor productivity of winter wheat under optimized fertilization in the Yangtze Valley (Sichuan, Yunnan, Guizhou, Chongqing, Hubei, Anhui, Jiangsu, Zhejiang and Shanghai).【Result】For all the data, the average yield of winter wheat in the Yangtze Valley under optimal fertilization treatment was 6.6 t·hm^-2, of these, Anhui province had the highest average yield with 7.3 t·hm^-2, and the lowest average yield with 3.6 t·hm^-2 in Chongqing. The average yield response of N, P and K fertilizer application were 2.3, 0.9 and 0.6 t·hm^-2, respectively, but there were significant differences among provinces. The average relative yield of N, P and K were 0.6, 0.8 and 0.9, respectively. The average agronomic efficiency of N, P and K were 12.6, 11.6 and 7.7 kg·kg^-1, respectively, and the average partial productivity were 34.0, 78.9 and 73.4 kg·kg^-1, respectively. As compared to farmers’ practices, the optimized fertilization treatment increased wheat yield by 0.5 t·hm^-2with an increase by 8.8%, increased agronomic efficiency of N, P and K fertilizer application by 41.1%, 121.1% and 84.6%, and partial factor productivity by 42.4%, 23.5% and 25.4%, respectively. 【Conclusion】Optimized fertilization had positive effect on the yield and nutrient use efficiency of winter wheat in the Yangtze Valley, but there were certain differences among provinces and great variation within provinces. There was the lower yield response in some provinces, such as Sichuan, Yunnan, Hubei and Jiangsu, indicated that had the higher soil nutrient supply in these regions. Therefore, optimal nutrient management should be formulated according to local conditions. By analyzing the parameters of wheat yield response and nutrient use efficiency under optimal nutrient management measured in the Yangtze Valley, nitrogen could be identified as the first limiting factor of wheat yield.

Status of Soil Fertility in Main Grape Producing Areas of China

LI BaoXin,YANG LiPing,LU YanLi,SHI XiaoXin,DU GuoQiang

Scientia Agricultura Sinica. 2020, 53(17): 3553-3566. doi:10.3864/j.issn.0578-1752.2020.17.012

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【Objective】The lagged soil fertility research and lacked scientific fertilization instruction in vineyards have gradually become the focus of restricting the sustainable development of the grape industry in China. Soil fertility status was investigated to formulate a suitable fertilization strategy so as to promote the healthy and sustainable development of grape industry in China. 【Method】1 100 soil samples were collected from the vineyards located in the five main grape producing areas in China (Northeast cold climate producing areas, North China and Bohai Bay producing areas, Qinling-Huaihe River subtropical producing areas, Northwest and Loess Plateau producing areas, Yunnan-Guizhou Plateau and Western Sichuan high altitude producing areas) respectively in 2018, and measured soil pH, organic matter, and available macro, medium and microelements content respectively. The status of soil fertility and its spatial distribution characteristics in the main grape producing areas of China were maped out by ArcGIS. 【Result】It showed that pH value range of soil in the main grape producing areas was 2.9-9.6, neutral soil was only 11.7% in selected soil samples in main grape producing areas of China; The average content of soil organic matter were 11.42 g·kg^-1 (deficient), and the proportion of soil with organic matter deficiency was 78.8%; The average contents of the nutrients were available N 77.8 mg·kg^-1 (medium), available P 97.2 mg·kg^-1 (rich), available K 214.7 mg·kg^-1 (medium), available Ca 1 670.8 mg·kg^-1 (rich), available Mg 299.0 mg·kg^-1 (rich), available S 72.5 mg·kg^-1 (extremely rich), available Fe 83.9 mg·kg^-1 (rich), available Cu 5.8 mg·kg^-1 (extremely rich), available Mn 16.1 mg·kg^-1 (rich), available Zn 6.5 mg·kg^-1 (rich), available B 2.86 mg·kg^-1 (rich). 【Conclusion】There was small proportion of soil with neutral pH value, which was suitable for grape growth in the main grape producing areas of China, and the area ratio is only 11.7%. Organic matter content is 11.42 g·kg^-1, and 78.8% of soil area with organic matter was at a deficient level. The content of available N (77.8 mg·kg^-1) and available K (214.7 mg·kg^-1) was insufficient, but the content of available P (97.2 mg·kg^-1) was at a very rich level. The content of available Ca, available Mg and available S was at a rich level, however, the content of available Ca and available Mg varies greatly between different regions. The micro element of available Cu (5.8 mg·kg^-1) was at a extremely rich level, the content of available Fe, Mn, Zn, and B was at rich level, but nutrient deficiencies still exist in some areas. Regional formula fertilization can be carried out according to the status of soil nutrients, so as to make reasonable supplement of macro, medium and micro elements.

Application of Fulvic Acid and Phosphorus Fertilizer on Tomato Growth, Development, and Phosphorus Utilization in Neutral and Alkaline Soil

ZHANG LiLi,SHI QingHua,GONG Biao

Scientia Agricultura Sinica. 2020, 53(17): 3567-3575. doi:10.3864/j.issn.0578-1752.2020.17.013

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【Objective】The phosphorus uptake and utilization of plants are reduced in alkaline soil. Fulvic acid (FA) application has effect on activating the hard-soluble phosphorus in soil. This study used FA to improve the utilization rate of phosphorus fertilizer in alkaline soil, so as to provide a new approach to reduce phosphorus fertilizer application. 【Method】The tomato (Solanum lycopersicum L.) cultivar of Luo La was used as plant material, which were cultivated in the big pots with two kinds soil. Two levels of soil pH (6.5 and 8.0) with four levels of phosphorus (P₂O₅) fertilizer application (0, 0.1325, 0.265 and 0.53 g·kg^-1, separately marked as 0% P, 25% P, 50% P and 100% P), with or without 0.08 g·kg^-1 FA were set in this experiment. Then, it was studied the effects of 0.08 g·kg^-1 FA and phosphorus fertilizer on plant growth, yield, fruit quality and phosphorus utilization in neutral and alkaline soil. 【Result】The tomato plant growth, yield, phosphorus concentrations, phosphorus uptake and fertilizer contribution were significantly reduced in alkaline soil. However, the tomato fruit quality was improved in alkaline soil. The tomato plant growth, yield and fruit quality, as well as phosphorus concentrations, phosphorus uptake and fertilizer contribution were also reduced by reducing phosphorus application. However, the phosphorus fertilizer utilization was improved in lower phosphorus levels. Application of FA had significant effects on improving phosphorus uptake and concentrations, which promoted the plant growth, yield and quality formation. Application of FA had little effect on phosphorus fertilizer utilization, which reduced fertilizer contribution in neutral soil. However, the phosphorus fertilizer utilization and fertilizer contribution were significantly enhanced by FA treatment in alkaline soil. In addition, application of FA increased levels of tomato growth and yield under 50% P treatment to that under 100% P treatment, while this enhanced effect was more significant in high pH soil. Application of FA to 100% P treatment had significant effects on improving tomato plant growth, yield in alkaline soil, which showed similar effects on that in 0% P without FA treatment in neutral soil.【Conclusion】Application of FA could improve phosphorus utilization, tomato plant growth, yield and fruit quality, which hit the mark of reducing phosphorus fertilizer application. Additionally, phosphorus fertilizer with FA application had mitigative effect of tomato growth and yield inhibition in alkaline soil.

Optimum Content of Mineral Elements in the Leaves of Duweiwendan Pomelo (Citrus grandis (L.) Osbeck. cv. Duweiwendan)

WANG XianDa, FAN GuoCheng, LI Jian

Scientia Agricultura Sinica. 2020, 53(17): 3576-3586. doi:10.3864/j.issn.0578-1752.2020.17.014

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【Objective】The aim of this study was to research and construct a diagnosis system for mineral nutrients in the leaves of Duweiwendan Pomelo (Citrus grandis (L.) Osbeck. cv. Duweiwendan), so as to deduce a shared optimization method for the mineral nutrition balance diagnosis and recommendation integrated system (BDRIS).【Method】From 2016 to 2018, 20 representative orchards with different managers were selected in the production area of Xianyou county in Fujian Province. 200 individual leaf samples and 8 660 single fruit samples were collected, and 48 pairs of heteromorphic fruits (H-FSI normal fruit and L-FSI cracking fruit) with the same axillary growth were collected, and the contents of N, P, K, CA, Mg, Cu, Zn, Fe, Mn, B, Mo and S elements in leaves and pericarps were measured. The internal and external quality of fruits and the width of top crack were also measured. Based on the difference between the total soluble solids (TSS) and the crack average index (FCAI) of the fruit, a BDRIS diagnosis system for major mineral elements and a diagnostic criterion for critical values of trace elements were constructed.【Result】According to the difference analysis of TSS, rich N, P, K and lack of Mo significantly reduced TSS. According to the FCAI12 difference analysis, except that the suspected B toxicity excess (>177 mg·kg^-1) significantly increased the fruit cracking, no significant causal relationship was found between other mineral elements and the fruit cracking, but no evidence of fruit cracking caused by B toxin was found; in addition, no significant difference was found in the paired comparative analysis of the elements in pericarp of twin shaped fruit. The distribution of N, P, K, CA, and Mg elements in the selected samples met the requirements of BDRIS modeling, P (normal) ≥0.12. According to BDRIS construction, it was found that the tree age was significantly negatively correlated with BDRIS index, and significantly positively correlated with TSS, that is to say, with the growth of tree age, the mineral nutrition of tree body tended to the origin of nutrition balance, and the fruit quality was also better, which gave a new explanation of the common sense of fruit tree cultivation that “the older the tree is, the sweeter the fruit will be”. BDRIS had no significant correlation with FCAI12; by correlation method, only when K was in morbid excess could thicken the peel, so as to reduce the cracking, which had no practical significance in correcting fruit cracking in production. It was suggested that the diagnostic standard of critical value of mineral elements in leaves should be “elements (over)”: N (<2.29%; 2.41%- 2.87%; >2.99%), P (<0.09%; 0.10%-0.14%; >0.15%), K(<1.17%; 1.39%-2.24%; >2.46%), Ca (<1.74%; 2.26%-4.21%; >4.72%), Mg (<0.20%; 0.24%-0.41%; >0.46%), Cu (<4 mg·kg ^-1; 6-25 mg·kg^-1; >30 mg·kg^-1), Zn (24-40 mg·kg^-1), Fe (60-140 mg·kg^-1), Mn (25-140 mg·kg^-1), B (<15 mg·kg^-1; 30-65 mg·kg^-1; >150 mg·kg^-1), Mo (<0.05 mg·kg^-1; 0.1-1.0 mg·kg^-1), and S (0.2%-0.4%). The shared optimization algorithm of BDRIS diagnosis parameters Mean, Std and r was provided to effectively expand the application scope of BDRIS.【Conclusion】In this study, the BDRIS diagnosis system for major mineral elements and the diagnostic criterion for critical values of trace elements were established based on the high-yield and high-quality population. In addition, there was no significant correlation between fruit cracking and the plant mineral nutrition.

Residue Behavior and Dietary Intake Risk Assessment of Imidaclothiz in Pakchoi (Brassica chinensis L.)

LI XiaoBei,ZHAO XiaoYan,LI JianYing,CHEN Lei,ZHOU ChangYan,HE XiangWei

Scientia Agricultura Sinica. 2020, 53(17): 3587-3596. doi:10.3864/j.issn.0578-1752.2020.17.015

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【Objective】The objective of the experiment was to reveal the residue behavior of imidaclothiz in pakchoi (Brassica chinensis L.), so as to provide a scientific basis for its safety utilization.【Method】Field experiments of 10 % imidaclothiz wettable powder in pakchoi under open field and greenhouse conditions were carried out in winter (between November and December) of 2018 and summer (between July and August) of 2019 at Shanghai. In dissipation experiments, the dosage of imidaclothiz was 90 g (a.i.)·hm^-2(1.5 times recommended dosage) with one-time spray, and the treated samples were collected randomly from several points of each plot at 2 h, 1, 2, 3, 4, 5, 7, 10, 14, 21, and 30 days after spraying of the pesticide to detect the residual concentration. For the study of final residue of imidaclothiz in pakchoi, imidaclothiz was sprayed for 2-3 times at an internal of 7 days at the recommended dosage (60 g (a.i.)·hm^-2) and 1.5 times recommended dosage (90 g (a.i.)·hm^-2), and the treated samples were collected randomly at 3, 5, and 7 days after the final processing to detect the residual concentration. The QuEChERS method coupled with ultra-high performance liquid chromatography-tandem mass spectrum (UPLC-MS/MS) was used to determine imidaclothiz in pakchoi. Dietary intake risk assessments were processed based on the maximal concentration, acceptable daily intake (ADI) of imidaclothiz, and daily consumption of pakchoi. The people involved in the experiment were divided into 8 classes, including underage male and female (subdivided into 3-6 years old infants and 7- 19 years old teenagers), as well as adult male and female (subdivided into 20-59 years old adults and 60-69 years old elder crowed).【Result】The limit of detection (LOD) of imidaclothiz was 0.0002 mg·kg^-1, and the limit of quantitation (LOQ) was 0.01 mg·kg^-1. Recoveries of imidaclothiz in pakchoi ranged from 77.2% to 87.9% at 0.01, 0.10 and 1.0 mg·kg^-1spiked levels, respectively, and the relative standard deviations (RSDs) were in the range of 2.5%-3.0%. The developed analytical method was suitable for the determination of imidaclothiz in pakchoi. Field experiments showed that the dissipation dynamics of imidaclothiz sprayed at the dosage of 90 g (a.i.)·hm^-2in pakchoi exhibited a first-order kinetic decline. The regression equation of imidaclothiz in winter greenhouse, summer greenhouse and summer open fields were C=0.8476e^-0.158^t, C=1.6558e^-0.212^t and C=4.3069e^-1.197^t, and their half-lives were 4.39, 3.27 and 0.58 days, respectively. Both existing time and plant conditions had significant correlation with degradation efficiency of imidaclothiz in pakchoi (P<0.05). The maximal concentrations of imidaclothiz in pakchoi under winter greenhouse were all below 0.5 mg·kg^-1 at 7 days after the final processing, when it was sprayed for 2-3 times at an internal of 7 days at the dosage of 60 or 90 g (a.i.)·hm^-2 in pakchoi, while it just required 3 days to decline below 0.5 mg·kg^-1 for those treated in summer in the same place, regardless of greenhouse and open fields. The final residue concentrations had positive correlation with spraying dosage, but no relationship with spraying number (P>0.05). The risk assessments showed that hazard quotients (HQs) of imidaclothiz for different groups consuming pakchoi were far below 1, and the maximum HQ was 0.2196.【Conclusion】Imidaclothiz was a kind of easily degradable pesticide, and its degradation rate was significantly higher in summer than winter, as well as higher in open fields than greenhouse. The dietary exposure of imidaclothiz only by pakchoi’s consumption was at a relatively low level to the ordinary resident of China. Generally, it’s effective to use imidaclothiz as the pest control method for pakchoi, accompanied with recommended dosage (45-60 g (a.i.)·hm^-2) and appropriate pre- harvest intervals (7 days in winter and 3 days in summer).

Screening and Analysis of Follicular Development Related Genes in Goat

ZHAO YuanYuan,LI PengFei,XU QinZhi,AN QingMing,MENG JinZhu

Scientia Agricultura Sinica. 2020, 53(17): 3597-3605. doi:10.3864/j.issn.0578-1752.2020.17.016

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【Background】 Dominant follicles (DF) and subordinate follicles (SF) were the most important two stages in the follicle development in the first follicle wave of goat. With further development of follicles, DF may eventually develop into mature follicles until ovulation, and SF will move towards atresia, while apoptosis of granulosa cells is the key factor leading to follicular atresia. However, the molecular mechanisms of promoting follicle dominance or causing its atresia are still unclear. 【Objective】This study was aimed to screen the key genes affecting follicular development and provide a theoretical basis for further exploring the regulation mechanism of follicular development by high-throughput sequencing of DF and SF granulosa cells in the first follicular wave of goat.【Method】Ten healthy Guizhou white goats were selected (1 year old), and the prostaglandin F_2α were injected respectively for estrus synchronization. B-type ultrasonography was used to detect the follicle growth situation, and then all of the goats were slaughtered when estrus had appeared for three days. DF (4.5-6 mm in diameter) and SF (3-4.5 mm in diameter) were obtained, granulosa cells were separated in the first follicle wave, respectively. Total RNA were extracted, and libraries were constructed and sequenced by Illumina Hiseq 2500 platform. FastQC was used to evaluate the quality of raw reads sequenced and filter them to obtain clean reads with high quality. Trinity was used to assemble clean reads from scratch to obtain unigenes. mRNA was obtained by comparing unigenes with goat RefSeq database using CLC Genomics Workbench. DESeq2 software was used to analyze the differential expression of the obtained mRNA. The goseq and kobas software were used for GO analysis and KEGG signal pathway analysis. Finally, qRT-PCR was used to verify the selected key genes that might affect the follicle development. 【Result】After the raw reads which obtained by sequencing were filtered, 43 217 934 clean reads were obtained from DF granulosa cells, accounting for 95.19% of raw reads. 40 766 348 clean reads were obtained from SF granulosa cells, accounting for 95.35% of raw reads. When the unigenes were compared with the RefSeq database of goat, a total of 33 896 annotated transcripts were obtained. Setting FPKM>1 and q value<0.05, a total of 13 644 genes were obtained in two types of follicle granulosa cells. By setting parameters: FPKM≥1, SF-FPKM/DF-FPKM>1, P<0.05, 695 differentially expressed mRNAs were obtained, of which 233 were significantly up-regulated and 462 were down-regulated in SF granulosa cells. GO functional enrichment analysis was performed on 695 differentially expressed mRNAs and concentrated in 42 groups of three major categories: biological processes accounted for 47.6%, cellular components 47.6%, and molecular functions 4.8%. KEGG signaling pathway analysis revealed 20 pathways, among which ribosome pathway related genes were most significantly enriched. Six genes that might be closely related to follicle development in goats were screened out, among which PRLR, PTX3 and RGN were up-regulated in subordinate follicles granulosa cells. DKK3, ALDH1A2 and RARRES1 were down-regulated. qRT-PCR showed that the expression trend of PRLR, RGN, DKK3, ALDH1A2 and RARRES1 was consistent with high-throughput sequencing results, and the expression level of RGN in SF granulosa cells was significantly higher than that of dominant follicles (P<0.01). The expression levels of DKK3, ALDH1A2 and RARRES1 in dominant follicles granulosa cells were significantly higher than subordinate follicles (P<0.01).【Conclusion】DKK3, ALDH1A2, RARRES1 and RGN had extremely significant differences in the expression levels of dominant follicles and subordinate follicles. It was speculated that those genes might promote the predominance of follicles or lead to atresia during the follicle development of goat, which was of great significance to further explore the regulation mechanism of follicular development.

Comparative Analysis of MicroRNAs and Corresponding Target mRNAs in Ascosphaera apis Mycelium and Spore

CHEN HuaZhi,ZHU ZhiWei,JIANG HaiBin,WANG Jie,FAN YuanChan,FAN XiaoXue,WAN JieQi,LU JiaXuan,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,CHEN DaFu,GUO Rui

Scientia Agricultura Sinica. 2020, 53(17): 3606-3619. doi:10.3864/j.issn.0578-1752.2020.17.017

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【Objective】Ascosphaera apis exclusively infects honeybee larvae, resulting in chalkbrood disease. The objective of this study is to clarify the differences of number, structure and expression pattern of miRNAs between A. apis mycelium and spore based on deep sequencing and comparative analysis of purified mycelia (AaM) and spores (AaS) using small RNA-seq (sRNA-seq) and bioinformatics, and reveal the potential relationship between common miRNAs, specific miRNAs, differentially expressed miRNAs (DEmiRNAs) and their target mRNAs and the growth and development of mycelium and spore as well as pathogenesis of A. apis.【Method】The pure culture of A. apis was gained under lab condition. AaM and AaS were respectively sequenced using sRNA-seq technology. Clean tags were obtained after filtration and quality control of raw reads. Common miRNAs and specific miRNAs in AaM and AaS were screened out using Venn analysis. DEmiRNAs in the AaM vs AaS comparison group were filtered out following the criteria of P≤0.05 and |log2 fold change|≥1. Target mRNAs of common miRNAs, specific miRNAs and DEmiRNAs were predicted using related bioinformatic software. Target mRNAs mentioned above were respectively annotated to GO database and KEGG database. The regulatory network of DEmiRNAs and target mRNAs was constructed on basis of target binding relationship, followed by visualization with Cytoscape. RT-qPCR was conducted to verify the reliability of the sequencing data.【Result】In total, 12 982 320 and 12 708 832 raw reads were produced from AaM and AaS, and after strict quality control, 10 800 101 and 9 888 848 clean tags were gained, respectively. The length of specific miRNAs in AaM was distributed among 18-26 nt, while that in AaS was distributed among 18-24 nt. Additionally, most of the miRNAs were distributed in 18 nt. MiRNAs with the first base U in both AaM and AaS were the most abundant. MiRNAs with the highest expression levels in both AaM and AaS were miR6478-x, miR10516-x and miR482-x. These common miRNAs could target 5 946 mRNAs, while specific miRNAs in AaM and AaS could bind to 6 141 and 6 346 mRNAs, respectively. Targets of common miRNAs were annotated to 42 functional terms such as metabolism process, cellular process and catalytic activity, and 120 pathways including translation, carbohydrate metabolism and energy metabolism. In addition, a total of 93 DEmiRNAs were identified in AaM vs AaS comparison group, targeting 6 090 mRNAs annotated to 38 functional terms and 120 pathways. Moreover, complicated regulatory networks were formed between DEmiRNAs and target mRNAs, with miR-4968-y located in the center and linked to as many as 118 mRNAs. RT-qPCR result demonstrated the expression trend of five DEmiRNAs was consistent with that in the sequencing result, confirming the reliability of our sequencing data.【Conclusion】The structures of miRNAs in A. apis mycelium and spore were similar, whereas their expression patterns were obviously different; mycelium and spore may specifically and differentially express part of miRNAs to regulate their growth, development and reproduction.

Estimation of Genetic Parameters of Body Weight and Egg Number Traits of Lueyang Black-Boned Chicken

DANG LiPing,ZHOU WenXin,LIU RuiFang,BAI Yun,WANG ZhePeng

Scientia Agricultura Sinica. 2020, 53(17): 3620-3628. doi:10.3864/j.issn.0578-1752.2020.17.018

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【Objective】Lueyang black-boned chicken (LBC) is an indigenous breed originating from Shaanxi province of China. The breed has large adult body weight, high meat quality, abundant contents of amino acids in meat and excellent adaptation to the free-range system in the forest. But the breed has poor performance of growth and egg production. We measured body weight (BW) and egg number (EN) of LBC at different ages. Based on these phenotypic data we evaluated genetic parameters of BW and EN traits. The aim of this study is to elucidate effect of genetic factors on these traits and genetic relationship among them, thus forming a theory foundation to breeding programs of LBC.【Method】BWs at 6, 10, 14, 20 weeks of age and first egg of 799 cocks and 804 hens from 71 half-sibling families of generation 1 of LBC breeding population and individual ENs from first egg to 31, 35 and 40 weeks of age were measured. Data lower than 1^st quantile-1.5×interquantile range and higher than 3^rd quantile + 1.5×interquantile range was deleted prior to statistical analysis. Errors in sex records were corrected. Heritabilities were estimated using a univariate animal model where sex was designed as the fix effect, and additive genetic effect was designed as random effect. Genetic correlations among traits were estimated using a bivariate animal model. Inverse gamma distributions were used as the prior distributions for variance components of random and residual effects. Posterior distributions of variance components were obtained based on 1 000 posterior estimates which were produced using Bayesian algorithm by running 130 000 iterations of which the first 30 000 were discarded in ‘burn-in’ period and the rest was sampled every 100 iterations. Heritabilities and genetic correlations were calculated according to posterior variance components. Phenotypic correlation among traits was calculated using chart.Correlation command in PerformanceAnalytics library of R.【Result】BWs of LBC cocks were (0.56±0.07), (1.07±0.13), (1.56±0.19), (1.97±0.21) kg and BWs of hens were (0.47±0.06), (0.86±0.12), (1.18±0.16), (1.48±0.19) and (1.68±0.23) kg at 6, 10, 14, 20 weeks and first egg. ENs were 31.2±11.5, 42.5±16.7 and 54.7±20.4 at 31, 35 and 40 weeks. Heritabilities of BWs at 6, 10, 14, 20 and first egg that were 0.74, 0.76, 0.63, 0.54 and 0.52 generally decreased with increasing age. Ninety-five percent confidence interval of BW heritabilities ranged from 0.25 to 0.33. Heritabilities of ENs at 31, 35 and 40 weeks were 0.27, 0.25 and 0.26 with 95% confidence interval varying between 0.35 and 0.42. Genetic correlation among 6, 10 and 14 week BWs were moderate to high (0.52-0.68). But BWs at 6 week (0.21-0.52) and first egg (0.21-0.46) showed relatively week genetic relationship with other BWs. Ninety-five percent confidence interval of genetic correlation coefficients among BW traits ranged from 0.13 to 0.34. Genetic correlation coefficients among EN traits approached to 1 with 95% confidence interval ranging from 0.03 to 0.06. BWs and ENs did not show any significant genetic relationship. On the temporal dimension phenotypic correlation coefficients varied between 0.43 and 0.90 (P<0.001) for BW traits and between 0.79 and 0.94 (P<0.001) for EN traits. There was weak (0.023-0.15) positive correlation between BWs at 6-20 weeks and ENs. But the BW at first egg showed weak negative correlation (-0.17-0.14) with ENs. 【Conclusion】This represents the first study specifically estimating genetic parameters for LBC. The data shows that BW traits of LBC are predominately determined by genetic factors. In contrast, ENs are more influenced by environmental factors. Results from genetic correlation analysis elucidated the genetic relationship between early and late traits, and between growth and reproduction in LBC, which provide a solid foundation for early selection and multiple-trait breeding programs. High heritabilities showed by BWs suggest that the phenotypic selection of BWs could significantly increase the growth rate of LBC. However it might be more reasonable to breed pure lines and take advantage of heterosis to increase egg yield of LBS. Genetic independence between BWs and ENs suggests that it is feasible to simultaneously improve two traits in the LBC.

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