Scientia Agricultura Sinica

Genome-Wide Association Study and Candidate Gene Mining of Tillering Number in Japonica Rice

ZHANG JiFeng,LIU HuaDong,WANG JingGuo,LIU HuaLong,SUN Jian,YANG LuoMiao,JIA Yan,WU WenShen,ZHENG HongLiang,ZOU DeTang

Scientia Agricultura Sinica. 2020, 53(16): 3205-3213. doi:10.3864/j.issn.0578-1752.2020.16.001

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【Objective】Genome-wide association study (GWAS) was used to detect SNP loci that were significantly related to tillering number in japonica rice, and to screen candidate genes affecting tillering number 【Method】This study used 295 japonica rice varieties from around the world, in 2018 and 2019 survey from the tillering stage of rice tillering number, the combination of high throughput sequencing weight gain high quality 788396 polymorphism, SNP, using TASSEL 5.0 software genome-wide association analysis of MLM model, using the GEC software to calculate the number of effective independent SNPS for the determination of threshold value, determine the significance of SNP markers associated with target traits. Based on the peak SNP detected in two years and the LD attenuation distance of each chromosome in rice, the major QTLs for co-localization of biennial number were determined, and the non-synonymous SNPs and promoter regions of all gene exon regions in the QTL interval were further extracted. SNPs were subjected to haplotype analysis, and then combined with gene annotation to screen candidate genes affecting japonica rice tillering number. 【Result】 The tillering number of 295 japonica rice varieties were basically the same in 2018 and 2019, and they all had a large phenotypic distribution. Under the threshold of P < 5.46 × 10^-6, three QTLs (qTiller8, qTiller9 and qTiller10) related to tillering number of Japonica rice were identified on chromosomes 8, 9 and 10 by genome-wide association analysis. qTiller9 jointly detected that the contribution rate of phenotype in 2018 and 2019 was 11.86% and 10.61%, respectively. qtiller8 and qtiller10 were only detected in 2018, and the contribution rate of phenotype was 10.61% The rates were 9.36% and 9.10%, respectively. Haplotype analysis of all 15 genes in qTiller9 interval showed that there were 6 genes in qTiller9 interval (LOC_Os09g25090, LOC_Os09g25100, LOC_Os09g25150, LOC_Os09g25190, LOC_Os09g25200 and LOC_Os09g25220). LOC_Os09g25090 was divided into two haplotypes by promoter SNP, and the tillering number of hap2 (TAA) was significantly higher than that of hap1 (AGG). LOC_Os09g25100 was divided into two haplotypes by non-synonymy mutation SNP, and hap2 (GAGA) had a significantly higher tillering number than hap1 (AGCC). LOC_Os09g25150 was divided into two haplotypes by non-synonymy mutation SNP, and the tillering number of hap2 (ATG) was significantly higher than that of hap1 (GCC). LOC_Os09g25190 was divided into two haplotypes by promoter SNP and the tillering number of hap2 (GCATCGCATCGACGCCGA) was significantly higher than that of hap1 (ATGCTGATGAAGTCATCC). LOC_Os09g25200 was divided into two haplotypes by non-synonymic mutant SNP and the tillering number of hap2 (TAG) was significantly higher than that of hap1 (AGA). LOC_Os09g25220 was divided into two haplotypes by non-synonymic mutant SNP, and the tillering number of hap1 (GG) was significantly higher than that of hap2 (AA).Combined with gene annotation, it was found that LOC_Os09g25090 and LOC_Os09g25100 both predicted the encoding of calcineurin dependent protein kinases and were Ca²⁺ sensors necessary for abscisic acid (ABA) expression. Previous studies have shown that ABA can affect both tillering number and branch number of Arabidopsis. Therefore, LOC_Os09g25090 and LOC_Os09g25100 are candidate genes affecting the tillering number in japonica rice. 【Conclusion】LOC_Os09g25090 and LOC_Os09g25100 were screened as candidate genes affecting the tiller number of japonica rice.

Screening of Broomcorn Millet Varieties Tolerant to Low Nitrogen Stress and the Comprehensive Evaluation of Their Agronomic Traits

CHEN Ling,WANG JunJie,WANG HaiGang,CAO XiaoNing,LIU SiChen,TIAN Xiang,QIN HuiBin,QIAO ZhiJun

Scientia Agricultura Sinica. 2020, 53(16): 3214-3224. doi:10.3864/j.issn.0578-1752.2020.16.002

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【Objective】The objective of this study is to find out the evaluation methods of low-nitrogen-tolerant broomcorn millet varieties, to screen the genotypes and identification indexes, and to provide a theoretical basis for the breeding of the low-nitrogen-tolerant varieties and understanding the physiological mechanism of the tolerance to low nitrogen stress.【Method】In the field experiment, 100 broomcorn millet varieties from domestic and foreign places were used for the treatment of low nitrogen stress (0 pure nitrogen) and normal nitrogen application (150 kg·hm^-2 pure nitrogen). Eleven indexes, including nine major agronomic traits (the plant height, stem diameter, number of main-stem nodes, panicle length, straw weight, panicle weight per plant, grain weight per plant, 1000-grain weight and leaf area), nitrogen content and nitrogen absorption were measured for two consecutive years. The low-nitrogen-tolerant index of each indicator was assessed by the method of membership function, and their comprehensive ability of low nitrogen tolerance was evaluated by principal component analysis, regression analysis and cluster analysis.【Result】There were significant differences in all eleven indexes, including plant height, stem diameter, number of main -stem nodes, panicle length, straw weight, panicle weight per plant, grain weight per plant, 1000-grain weight, leaf area, nitrogen content and nitrogen absorption of the tested varieties at different nitrogen levels. Under low nitrogen stress, the growth, biomass accumulation and nitrogen absorption of broomcorn millet were inhibited. The value of each agronomic trait decreased significantly, and the range of variation was reduced. The order of reduction range was that leaf area > straw weight > grain weight per plant > panicle weight per plant > stem diameter > number of main-stem nodes > panicle length > 1000-grain weight > plant height. The nitrogen content and the nitrogen absorption of different broomcorn millet seeds decreased and the decrease range of nitrogen absorption decrease was higher than that of nitrogen content. Under low nitrogen stress, the variation coefficients of plant height, stem diameter, number of main-stem nodes, panicle length, straw weight, panicle weight per plant and grain weight per plant in different broomcorn millet varieties were higher than those of normal nitrogen application levels. Under different nitrogen levels, the variation coefficient of nitrogen absorption in different broomcorn millet seeds was higher than that of nitrogen content. The variation coefficient of nitrogen absorption under low nitrogen stress was higher than that of normal nitrogen application. The principal component analysis was carried out on the low nitrogen tolerance index of 11 indicators, and the five principal components were selected. The cumulative variance contribution rate was 75.83%. The correlation between the low nitrogen tolerance index and the comprehensive evaluation value on the low nitrogen tolerance (D) of seven indicators (the plant height, panicle length, straw weight, panicle weight per plant, grain weight per plant, leaf area, nitrogen absorption) was highly significant. Among them, the correlation of panicle weight per plant, grain weight per plant and nitrogen absorption was higher, with the correlation coefficients 0.858, 0.812 and 0.812, respectively. According to the D-value, 100 varieties of broomcorn millet were classified into three types: Low nitrogen tolerance, intermediate and low nitrogen susceptible.【Conclusion】Through the analysis of the comprehensive characteristics of each trait index, it is concluded that the panicle weight per plant, straw weight, and nitrogen absorption can be used as the first choice for the evaluation of the ability of low nitrogen tolerance. Four varieties of Yumi 3, 2058, Yushu 1 and Yanshu 7 have the strongest tolerance to nitrogen-deficiency.

Creating a New-Type Brassica napus (AⁿA^rCⁿC^o) with High Drought-resistance Employing Hexaploid (AⁿAⁿCⁿCⁿC^oC^o) as a Bridge

WAN HuaFang,WEI Shuai,FENG YuXia,QIAN Wei

Scientia Agricultura Sinica. 2020, 53(16): 3225-3234. doi:10.3864/j.issn.0578-1752.2020.16.003

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【Objective】As one parent of Brassica napus, B. rapa has a wide range of variation, as well as excellent performance against drought or poor soil. Employing hexaploid as a bridge, the drought resistance of B. rapa is transferred to develop a new-type B. napus with excellent drought tolerance.【Method】The hexaploid (AⁿAⁿCⁿCⁿC^oC^o) was created via interspecific cross between B. napus (AⁿAⁿCⁿCⁿ) and B. oleracea (C^oC^o) via embryo rescue and chrosome doubling. Next, a new-type B. napus（AⁿA^rCⁿC^o）was developed by crossing the hexaploid with B. rapa (A^rA^r). Under the treatment with PEG-6000 solution to imitate drought stress at germination stage, the indices related to drought resistance were measured in the experimental materials, including natural B. napus, new-type B. napus and B.rapa. All the datum were analyzed with subordinate function method.【Result】 Using 11 hexaploid materials and 68 B. rapa materials as parents, 124 new-type B. napus lines were created, which showed intermediate morphology between hexaploid and B. rapa. The hexaploid used in the experiment all had 56 chrosomes and the new-type B. napus had 38 chromsomes. The pollen fertility in the hexaploid and the new-type B. napus was approximately 90% and 80%, respectively. Based on the results of principal components analysis and SSR analysis, 7 hexaploid, 59 new-type B. napus, 10 B. rapa and 20 natural B. napus were clustered into three groups, namely B.rapa, hexaploid and B. napus. The new-type B. napus and natural B. napus were in the same group, but they clearly separated each other. Based on the drought resistance-related indices, 200 g·L^-1 and 250 g·L^-1 PEG-6000 solution were used to imitate drought stress to B. rapa and new-type B. napus, respectively. Nine new-type B. napus were tested, but only three were more resistant than the control, Zhongshuang 11. Additionally, the resistance were positively associated to the corresponding parent B. rapa, which indicated that the three new-type B. napus have been transfered excellent drought resistance from B. rapa.【Conclusion】 Using B. rapa as a drought resistance donor and the hexaploid as a bridge, new-type B. napus lines with excellent drought tolerance were developed.

Research Progress on the Crop Growth Model CropGrow

ZHU Yan,TANG Liang,LIU LeiLei,LIU Bing,ZHANG XiaoHu,QIU XiaoLei,TIAN YongChao,CAO WeiXing

Scientia Agricultura Sinica. 2020, 53(16): 3235-3256. doi:10.3864/j.issn.0578-1752.2020.16.004

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Agricultural information technology is formed as the result of integrating information technology and agricultural science, and has further facilitated the rapid development of digital agriculture (DA) and smart agriculture (SA). As one of the core technologies of DA and SA, crop growth model can dynamically simulate crop growth and development processes and their relationships with climate condition, soil characteristics and management strategy, so as to overcome the limitation of the spatial-temporal characteristics of traditional research on agricultural production management. It can provide powerful quantitative tools for crop productivity prediction and early warning and impact evaluation under different conditions. Through over 20-years systematic and profound exploration and practicing in wheat and rice crops, and based on the workflow of “physiological mechanism analysis-model algorithm development-dynamic productivity prediction-quantitative effect assessment-simulation platform development”, our research team has been devoted to the development and application of crop simulation model CropGrow, by integrating the technologies of system analysis, dynamic modeling, virtual reality, scenario simulation, and decision support. Firstly, based on the system analysis method and dynamic modeling technology, the comprehensive and mechanistic crop growth model CropGrow has been developed, including the submodels of phasic development and phenology, organ development and population establishment, photosynthetic production and biomass accumulation, assimilate partitioning and yield/quality formation, nutrient dynamics, and water balance, along with three-dimensional morphological and visual submodels, which could digitalize and visualize the processes of crop growth and productivity formation under different conditions. Further, by coupling geographic information system (GIS) and remote sensing (RS), the model-based regional crop productivity prediction technology has been established. Then, based on the scenario analysis, the contributions of climate change, soil improvement, variety updating, and strategy optimization to regional crop production have been quantified, and applications extended to generation of suitable management plan, design of ideal cultivar, assessment of climate impact, evaluation of land use and decision-making of agricultural policy. Finally, based on the component-based programming technology, a model-based digital and visual crop growth simulation system and decision support platform has been developed by integrating the crop production database and crop model components, further realizing the comprehensive functions of data management, parameter optimization, growth simulation, remote sensing coupling, regional prediction, management strategy design, effect evaluation, safety early warning and product release. In the future, based on the improvement of agro-information database, additional efforts in crop modeling will be made toward enhancing prediction ability, quantifying gene effects, developing intelligent decision-making, and coupling multiple models, which will provide digital support for the prediction and early warning of food production, quantitative evaluation of scenario effects, decision-making on management strategy, and optimal design of new crop cultivars, thus facilitating the security of national food and development of digital agriculture.

Research and Development of the Intelligent Identification System of Agricultural Pests for Mobile Terminals

SHAO ZeZhong,YAO Qing,TANG Jian,LI HanQiong,YANG BaoJun,LÜ Jun,CHEN Yi

Scientia Agricultura Sinica. 2020, 53(16): 3257-3268. doi:10.3864/j.issn.0578-1752.2020.16.005

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【Objective】There are various species of pests in crop fields, and interspecific similarity and intraspecific difference are common in agricultural pests, which are easy to be confused. In this study, an intelligent system based on mobile terminals was developed to identify agricultural field pests. This system is an easy and intelligent tool of pest identification for peasants and pest forecasting technicians. 【Method】The intelligent identification system of agricultural field pests consists of a mobile client with a system APP, a server and a pest identification model based on deep learning. The application (APP) can be installed in mobile devices with Android system and includes user registration, pest information inquiry, pest automatic identification, pest location information and remote expert identification. The UI interface in this APP uses the style of bottom navigation bar, the information exchange between mobile client and server adopts HTTP protocol and the SDK of Baidu Android map is used to mark the geographic information of pests. The information of users and pests is saved in MySQL database. In the same training and testing sets, different convolutional neural network models were developed to identify agricultural pests. The results showed that the DenseNet121 model achieved the highest precision and lowest false alarm rate. The pest identification model based on DenseNet121 was installed in Alibaba Cloud remote server. When the server received the images from the mobile clients, the identification model was performed. The identification results were feed back to clients from the server. All images and results were saved in database for being traced back in future.【Result】When the users met unidentified pests in crop fields, the users could collect pest images and upload them to the server by the APP installed in mobile clients, such as mobile phone or PAD. The identification results and pest control information would be fed back to the mobile clients in 1-2 seconds. If the results were unsatisfied, the user could ask the expert to remotely identify pests. This system could identify 66 species of pests, and the average precision was 93.9% and false alarm rate was 8.2%. 【Conclusion】The intelligent identification system of agricultural pests could automatically identify the 66 species of agricultural pests. The system could inquire pest information, show the pest geographic information, and ask expert to remotely identify pests. This system is a tool for peasants and pest forecasting technicians to easily and accurately identify agricultural pests in crop fields. It can provide users the one-to-one pest control information and experts needn't go to crop fields for guiding peasants to manually identify pests. This system can save money and time.

Composition and Distribution Characteristics of Pathogens Causing Wheat Sharp Eyespot in Wheat and Maize Double Cropping System

ZHAO XuSheng,QI YongZhi,ZHEN WenChao

Scientia Agricultura Sinica. 2020, 53(16): 3269-3279. doi:10.3864/j.issn.0578-1752.2020.16.006

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【Objective】Wheat sharp eyespot (WSE) occurs seriously in wheat and maize double cropping system with straw returning to field in Hebei, Shandong and Henan provinces. The objective of this study is to determine the disease occurrence, pathogen population structure and the farmland distribution characteristics of dominant pathogen by extensive investigation, and to provide a basis for the WSE control.【Method】The random five-point sampling method was used to investigate the occurrence degree of WSE in wheat and maize double cropping system with straw returning to field in Hebei, Shandong and Henan provinces. The population characteristics of Rhizoctonia isolates were identified by nuclear staining, hyphal fusion reaction and rDNA sequence analysis. The pathogenicity of 120 R. cerealis strains was determined by pot experiment, and the distribution characteristics of R. cerealis in the field of wheat and maize double cropping system were analyzed by real-time fluorescent quantitative PCR.【Result】The occurrence degree of WSE from serious to light was in the order of Henan, Shandong and Hebei. There were 421 binucleate R. cerealis strains and 24 multinucleate R. sonalis strains collected from 72 collection sites of 11 different ecological regions in Hebei, Shandong and Henan provinces, which could be divided into four types: AG-D, AG-B (0), AG-2 and AG-5 fusion groups, accounting for 91.91%, 2.70%, 3.82% and 1.57% of the total number of tested strains, respectively. According to the disease index of Shixin 828, Jimai 22 and Zhoumai 22, the 120 tested strains could be divided into five pathogenic types: VT1, VT2, VT3, VT4 and VT5. The disease indexes of each type were 44.0, 39.4, 34.2, 29.6 and 27.2 in turn, respectively. Among them, most of the strains from the wheat region in Henan Province had the strongest pathogenicity, and those collected from the wheat region in Hebei Province was the weakest. During the whole growth stage of wheat, the content of R. cerealis increased first and then decreased in overground and root systems. The amount of R. cerealis in the shoot was the lowest at the three-leaf stage, and the highest at the rising stage, with the value of 3 774.60 ng DNA/g fresh tissue. The amount of R. cerealis in the roots reached the peak at the jointing stage. The growth rate of R. cerealis amount was the fastest from the three-leaf stage to the tillering stage. The increase of R. cerealis content in the aboveground parts and roots of wheat was up to 28.17 and 17.26 times, respectively. The amount of R. cerealis increased gradually in rhizosphere soil of wheat and in the shoots of maize. In the rhizosphere soil of maize, the content decreased first and then increased, and the lowest at the tasseling stage was 170.6 ng DNA/g dry soil.【Conclusion】The most serious degree of WSE occurs in wheat area in Henan Province, followed by Shandong and Hebei. The strong pathogenicity of dinucleus R. cerealis is the dominant pathogen in the three wheat areas, the amount of which increases the fastest from the three-leaf stage to the tillering stage, and the relative content in wheat straw is the highest and that in maize straw is the lowest. In the process of crushing returning-straw and at wheat seedling stage before the winter, the monitoring and control of WSE should be strengthened.

Identification of Co-Expression Genes Related to Endogenous Abscisic Acid in Response to the Stress of Sclerospora graminicola by WGCNA in Foxtail Millet

CHANG GuoRong,LI RenJian,ZHANG Qi,ZHANG YuMing,HAN YuanHuai,ZHANG BaoJun

Scientia Agricultura Sinica. 2020, 53(16): 3280-3293. doi:10.3864/j.issn.0578-1752.2020.16.007

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【Objective】Abscisic acid (ABA), as a stress hormone, plays an important role in plant growth and development, biological and abiotic stresses. The ABA receptor protein PYR/PYL/PCAR and SNF1-related protein kinase (SnRK2) are important regulatory factors that mediate ABA signaling. The objective of this study is to predict the regulatory roles of ABA and the key genes in its signaling pathway in foxtail millet (Setaria italica) downy mildew caused by Sclerospora graminicola, and to provide a reference for the research of endogenous ABA in S. italica in response to the infection of S. graminicola.【Method】S. italica variety Jingu 21 infected by S. graminicola was used for transcriptome sequencing and ABA content measurement, and the PYL and SnRK2 family genes in the ABA signaling pathway were identified and analyzed based on the whole genome sequence of S. italica. A weighted gene co-expression network analysis (WGCNA) was constructed using the transcriptome, and it was associated with the content of host endogenous ABA in the context of S. graminicola infection to predict the key genes in the interaction between S. italica and S. graminicola regulated by ABA and the PYL and SnRK2 genes in its downstream signaling. The candidate genes were validated by qRT-PCR.【Result】There were 11 PYL and 11 SnRK2 family genes in S. italica, which were relatively conservative in the Gramineae, and ABA responsive elements were predicted in the promoters of PYL and SnRK2 family genes. The endogenous ABA in the host accumulated at the first and second stages after S. graminicola infection, and its content (22.50 and 18.08 ng·mL^-1, respectively) was significantly higher than that of the control group. However, the content of ABA decreased at the third, fourth, and fifth stages, which was lower than that of the control group. For the WGCNA, a total of 34 gene co-expression modules were constructed by using 18 535 genes. The MEpaleturquoise and MEbrown modules were predicted as the core candidate modules through the association analysis of ABA content and PYL, SnRK2 family genes. GO function enrichment and module key gene mining revealed that one PYL family gene (Seita.1G030500), two SnRK2 family genes (Seita.2G394500 and Seita.3G03200), and three core genes (Seita.4G105600, Seita.6G218100, and Seita.9G138400) might be involved in the interaction between S. italica and S. graminicola during the regulation of ABA and its signal transduction. After comparing three predicted core genes with the reference genes in Oryza sativa and Arabidopsis thaliana databases, Seita.4G105600 was identified as the transducin/WD40 repeat-like superfamily protein, Seita.6G218100 as the WRKY57 transcription factor, and Seita.9G138400 as the TIFY transcription factor. qRT-PCR analysis showed that Seita.2G394500, Seita.4G105600, and Seita.6G218100 genes were up-regulated at the early stage of S. graminicola infection.【Conclusion】The ABA accumulates in S. italica infected by S. graminicola. One PYL family gene, two SnRK2 family genes, two transcription factor genes, and one WD40 family protein gene were predicted as the key genes related to the response to S. graminicola infection. qRT-PCR results showed that one SnRK2 gene, one WD40 family protein gene, and one WRKY57 transcription factor gene may play an important role in the response of ABA in S. italica to the infection of S. graminicola.

Spatiotemporal Variability Characteristics of Soil Fertility in Red Soil Paddy Region in the Past 35 Years—A Case Study of Jinxian County

WANG YuanPeng,HUANG Jing,SUN YuXiang,LIU KaiLou,ZHOU Hu,HAN TianFu,DU JiangXue,JIANG XianJun,CHEN Jin,ZHANG HuiMin

Scientia Agricultura Sinica. 2020, 53(16): 3294-3306. doi:10.3864/j.issn.0578-1752.2020.16.008

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【Objective】The aim of this study was to clarify the changes of soil nutrients and the current fertility level in the actual agricultural production in the typical county of red soil, which provided the basis for the improvement of red soil paddy soil. 【Method】Data of paddy field soil properties in Jinxian County of Jiangxi Province in 1982, 2008 and 2017 were obtained through data collection and field sampling analysis. Soil pH, organic matter, alkalide nitrogen, available phosphorus and available potassium were selected as soil comprehensive fertility evaluation factors. Firstly, the conventional statistics and differential analysis of various fertility factors in the three periods were carried out. Principal component analysis was used to find out the key factors of fertility differences in different periods and the weight was determined. The membership value of each fertility factor was obtained by membership function. The soil integrated fertility index was calculated by weight values and membership values. Finally, the spatial and temporal evolution characteristics of paddy field soil fertility in this region were explored based on GIS spatial distribution maps of soil fertility factors and integrated fertility index. 【Result】From 1982 to 2017, soil organic matter, alkalide nitrogen, available phosphorus and available potassium of paddy soil in Jinxian County increased in varying degrees, while soil pH value decreased. The average pH values of Jinxian County in the three periods of 1982, 2008 and 2017 were 5.9, 5.1 and 4.8, respectively, with an average annual decrease of 0.03 units. In the past 35 years, the overall decline rate of soil pH from west to southeast and northwest gradually decreased. In 2017, 99% of the paddy soil was at an acidic level (4.5-5.5). The average content of soil organic matter increased from 28.1 to 36.8 g·kg^-1 in 35 years. The average annual increase rates of soil organic matter from 1982 to 2008 and from 2008 to 2017 were 0.21 and 0.31 g·kg^-1, respectively. In 2017, the proportion of paddy fields with soil organic matter content between 30 and 40 g·kg^-1 reached 94%. From 1982 to 2017, the increasing rate of soil organic matter decreased gradually from northeast to southwest. From 1982 to 2017, the average content of available phosphorus in soil increased from 7.0 to 32.1 mg·kg^-1. In 2017, the paddy fields with soil available phosphorus content ranging from 20 to 40 mg·kg^-1 were dominant in Jinxian County, accounting for 75%. The accumulation of available potassium in paddy soils was slow from 1982 to 2017. The annual average increase rates of available potassium in paddy soils from 1982 to 2008 and from 2008 to 2017 were 0.58 and 0.53 mg·kg^-1, respectively. In 2017, the average content of available potassium in paddy soil in Jinxian County was 73.2 mg·kg^-1. The alkalide nitrogen in paddy soils increased first and then slowly in the two stages of 1982-2008 and 2008-2017. The growth rates of the two stages were 1.24 and 0.29 mg·kg^-1, respectively. From 1982 to 2017, the increase rate of soil alkalide nitrogen content in Jinxian County was higher in Southeast China and lower in Northwest China. The average values of the integrated fertility index of paddy soil in Jinxian County in 1982, 2008 and 2017 were 0.43, 0.50 and 0.55, respectively. The general scores of soil fertility factors in three periods were as follows: alkalide nitrogen>available phosphorus>pH>available potassium>organic matter (1982), pH>available phosphorus>available potassium>organic matter>alkalide nitrogen (2008), available potassium>available phosphorus>pH>alkalide nitrogen>organic matter (2017). 【Conclusion】 After 35 years of long-term cultivation, the soil fertility of rice fields in Jinxian County has been improved. At present, the paddy soil in Jinxian County still has problems such as excessive alkalide nitrogen, available potassium deficiency, and serious acidification of the soil. Soil alkalide nitrogen, pH and available potassium were the key factors for the spatial distribution of soil fertility in Jinxian County during the three periods of 1982, 2008 and 2017, respectively.

Effects of Continuous Application of Soil Amendments on Fluvo- Aquic Soil Fertility and Active Organic Carbon Components

ZHOU JiXiang,ZHANG He,YANG Jing,LI GuiHua,ZHANG JianFeng

Scientia Agricultura Sinica. 2020, 53(16): 3307-3318. doi:10.3864/j.issn.0578-1752.2020.16.009

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【Objective】Impacting of two soil amendments on the contents of active organic carbon components and soil carbon pool management index were studied from 2015 to 2018 in a sandy fluvo-aquic soil in Langfang City, Hebei Province under wheat-corn rotation. The purpose of the study was to provide a theoretical basis for the higher accumulation of organic carbon in sandy fluvo-aquic soil and for the improvement of soil quality. 【Method】 The experiment was carried out by a single factor randomized block design with four treatments: (1) chemical fertilizer only (CK), (2) CK+ organic amendment 15 t·hm^-2 (T1), (3) CK+ inorganic amendment 2.25 t·hm^-2(T2), and (4) CK+ organic amendment 15 t·hm^-2+ inorganic amendment 2.25 t·hm^-2 (T3). Soil organic matter content, total nitrogen, pH, available phosphorus and available potassium were measured to calculate soil integrated fertility index (IFI) by modified Nemero index method. Active organic carbon pools were also measured to calculate carbon pool management index (CPMI). Both IFI and CPMI were used to evaluate the soil amendments for the improvement of soil quality. 【Result】 The results showed that soil organic carbon (TOC) and IFI increased significantly under organic amendment comparing to CK, especially under the combination of organic with inorganic amendment (T3). The contents of active carbon pools increased under organic amendment, and was in the order of easily labile organic carbon (LOC)> dissolved organic carbon (DOC)>microbial biomass carbon (MBC). The ratio of active carbon pools to TOC decreased under organic amendment: the ratio of easily labile organic carbon to TOC （LOC/TOC） decreased significantly by 12.57% and 12.02% under T1 and T3, respectively; the ratio of microbial biomass carbon to TOC MBC/TOC decreased significantly by 12.84% and 12.30% under T1 and T3, respectively. Inorganic amendment (T2) had no effects on TOC and active carbon pools, therefore, the decreased ratios of active carbon pools to TOC mean that organic amendment increased stable TOC more than active carbon pools. The increase of CPMI and decrease of the ratio of soil carbon pools to TOC under organic amendment (T1 and T3) indicated that organic amendment resulted in the accumulation of organic carbon. The CPMI increased under the organic amendment. Principal component analysis showed that the organic amendment affected the composition and ratio of active carbon pools in the soil. 【Conclusion】 Continuous application of organic amendment significantly improved fertility of sandy fluvo-aquic soil, increased soil carbon pool management index and carbon storage capacity, and ultimately improved soil quality.

Impact of Soil Data with Different Precision on Water Quality and Flow Simulation

LI Ying,LEI QiuLiang,QIN LiHuan,ZHU AXing,LI XiaoHong,ZHAI LiMei,WANG HongYuan,WU ShuXia,YAN TieZhu,LI WenChao,HU WanLi,REN TianZhi,LIU HongBin

Scientia Agricultura Sinica. 2020, 53(16): 3319-3332. doi:10.3864/j.issn.0578-1752.2020.16.010

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【Background】 Watershed model is an important tool to study non-point source pollution, and the reliability of input data is an important factor to ensure the accuracy of the model estimation. Soil data, as one of the important input data of the watershed model, has a significant influence on the runoff process. The previous studies mainly focused on the water quantity and hydrological process, while the research on water quality needs more attention. Furthermore, the disagreements over appropriate resolution of the soil maps also existed in the previous studies. 【Objective】 The purpose of this study was to enrich the prior knowledge of modeling and to provide useful suggestion for data selection in watershed simulation. 【Method】 This paper employed the widely used SWAT (soil & water assessment tool) model as an example and simulated flow, sediment, TN (total nitrogen) and TP (total phosphorus) of Fengyu river basin by three types of soil data with different scales. The scales were used for soil data as follows: 1﹕50 000 (soil-1), 1﹕500 000 (soil-2) and 1﹕1 000 000 (soil-3). SWAT-CUP software was used to adjust parameters. Based on the above methods, the effects of soil data with different scales (1﹕50 000, 1﹕500 000 and 1﹕1 000 000) on HRU (hydrologic response unit) division, model parameters and water quality and quantity were studied. 【Result】 (1) Soil data with different scales had great influence on the result of the HRU division, and the sensitivity of the number of HRU division was related to the division threshold and soil map precision. (2) The calibration of model parameters could remarkably improve the simulation performance of the model. The simulation performance associated with simulated substance, and the highest precision soil map not had the best simulation performance. (3) The attributes of soil tended to be consistent with the increase of sub-watershed area. The variation of simulation results caused by different soils gradually tends to be steady with the increase of confluence area, and the calibration process could have a great effect when confluence area was small. 【Conclusion】 In summary, the precision of soil data should be selected according to the basin size and simulated substance, and the impact of spatial scale need to be considered.

Genetic Relationship and Structure Analysis of 15 Species of Malus Mill. Based on SNP Markers

GAO Yuan,WANG DaJiang,WANG Kun,CONG PeiHua,LI LianWen,PIAO JiCheng

Scientia Agricultura Sinica. 2020, 53(16): 3333-3343. doi:10.3864/j.issn.0578-1752.2020.16.011

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【Objective】 Based on SNP markers developed by high-throughput, the simplified genomic sequencing technology in the whole genome, the genetic relationship and population genetic structure of native germplasms of Malus Mill. in China were analyzed, which provided a theoretical basis for the origin, evolution and systematic classification of Malus Mill.【Method】Based on obtained SLAF tags, BWA software was used to locate them in the reference genome and to obtain the polymorphic SLAF tags. GATK and SAMtool were employed to develop SNPs in polymorphic SLAF tags and screened SNPs obtained by two methods as SNP markers data set. According to the integrity > 0.94 and the minor allele frequency (MAF) > 0.05, SNPs with polymorphism were screened. Based on the screening of polymorphic SNPs, the NJ (neighbor joining) algorithm of MEGA 7 was used to construct phylogenetic trees of different species of Malus Mill. The genetic structure was analyzed by using the software of admixture. Assuming that the number of clusters (k) was 1-15. The best K value was determined according to cross validation error rate, and the genetic structure among and within species of Malus Mill. was analyzed.【Result】427 accessions of Malus Mill. were sequenced by SLAF-seq, and 586 454 SLAF tags were obtained, including 463 612 polymorphic SLAF tags. 46 460 polymorphic single nucleotide (SNP) loci were obtained through sequence alignment analysis and screening. Phylogenetic tree of different species of Malus Mill. was constructed based on these SNP loci, and population structure was analyzed. In phylogenetic analysis, 15 species of Malus Mill. were divided into 4 groups, and K = 5 and K = 14 were key points in the genetic structure analysis. According to the results of two methods, 15 species of Malus Mill. could be divided into 4 basic groups, which were the group of Malus baccata, the group of Malus sieversii and a few accessions of Malus domestica subsp.chinensis, the group of Malus toringoides, Malus transitoria, Malus kansuensis, Malus komarovii, Malus yunnanensis and Malus ombrophila, and the group of Malus domestica subsp. chinensis, Malus robusta, Malus asiatica and Malus prunifolia. There were gene backgrounds of Malus sieversii and Malus baccata in some germplasms of Malus domestica subsp. chinensis, but there were also some germplasms of Malus domestica subsp. Chinensis, which could independently represent the group gene pool, and the gene pool was not involved in Malus sieversii, but closely related to Malus baccata, Malus asiatica and Malus prunifolia.【Conclusion】The rapid discovery of 46 460 polymorphic SNP markers covering the whole genome by SLAF technology could effectively study on the genetic relationship and structure within and among species of Malus Mill. in China, and provide references for identification and evaluation, genetic diversity, systematic classification, origin and evolution of germplasm resources of Malus Mill. 15 species of Malus Mill. could be divided into 4 basic groups, and the classification of wild and cultivated species of Malus Mill. was obvious. The genetic relationship between Malus domestica subsp. chinensis and other cultivated species was close.

Optimization of Embryo Rescue Technique and Production of Potential Seedless Grape Germplasm with Rosy Aroma

JIA ShanShan,LUO QiangWei,LI ShaSha,WANG YueJin

Scientia Agricultura Sinica. 2020, 53(16): 3344-3355. doi:10.3864/j.issn.0578-1752.2020.16.012

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【Objective】The aim of this study was to investigate the grape scent traits to provide important material basis for seedless aroma grape breeding. By exploring the parental genotype, embryo development morphology and growth regulator on embryo rescue efficiency, further optimizing the seedless × muscat scent combined embryo rescue system, and molecular marker technology was used to initially identify seedless traits of hybrid offspring.【Method】Headspace solid phase micro-extraction (HS-SPME) combined with gas chromatography-mass spectrometry technique was used to determine the content of aromatic substances in the grapes of 10 different muscat-scented and non-muscat scented varieties. Excellent aroma varieties were selected as parents, then combined with the previous research results of the group, 13 cross-combinations, including 6 female parents and 5 male parents, were used to obtain new grapevines with seedlessness and aroma. Under aseptic conditions, the ovules were cultured from young fruits in the dark for 8 weeks. The developed hybrid embryos were inoculated in a variety of embryo germination media, with different concentrations and different proportions of hormones to assess and improve the germination rate and germination seedling rate. After the embryo developed into plants, they were placed in the greenhouse for hardening, the survived F₁ generation was transplanted to the field. The molecular marker technology was used to select the seedless characters of F₁hybrid lines in the early stage. 【Result】Among 10 table grapes, it was the terpenes that determined the aroma of muscat, which could be detected in 8 muscat scent varieties, and the content of which ranged from 0.0246 to 1.3824. Based on the above evaluation, ‘Muscat of Alexandria’, ‘Shine-Muscat’ and ‘Muscat Hamburg’ were selected to create new seedless aroma germplasm because their high levels of terpenoids. The study showed 1 284 progeny were obtained by embryo rescue technique, and 697 of them survived after transplanting into field. Ruby Seedless and Flame Seedless with a higher seedling rate were suitable as female parent in embryo rescue. Muscat Hamburg and Shine-Muscat proved as the better male parents. Among them, the embryo development rate and seedling formation rate of Ruby seedless × Aishenmeigui were the highest, at 48.59% and 51.71%, respectively. The germination rate was the highest when embryos were inoculated into WPM as based medium, and the growth regulator (1.0 mg?L^-1 KT + 0.5 mg?L^-1 NAA + 1.0 mg?L^-1 ZT) was added, which was 11.33%, higher than the control which was 7.41%. Three molecular markers GLSP1-569, SCF27-2000 and SCC8-1018 were selected for detecting the cross parents and hybrid progenies. The results showed that 27 out of 101 hybrid strains had seedless genes. 【Conclusion】 Muscat of Alexandria and Sunshine-Muscat had a high scent content, and the embryo rescue efficiency was high after crossing with European grapes, which was a suitable scented parent material. Ruby Seedless and Flame Seedless was suitable as female parent. During embryo germination and culture, using WPM medium as the basic medium, adding appropriate concentrations of KT and ZT had a better effect of promoting root growth. The seedless traits of hybrid F₁ generation were detected by molecular markers, and the seedless rate was 26.73%. At present, few aroma markers have been developed, so the hybrid offspring obtained in this study could provide an important material basis for the further study of aroma gene markers.

Nano-Packaging Preservative Mechanism of Flammulina filiformis After Harvest Based on Mitochondrial Energy Status Pathways

MA Ning,WANG HeTong,FANG DongLu,ZHAO LiYan,YANG WenJian,PEI Fei,HU QiuHui

Scientia Agricultura Sinica. 2020, 53(16): 3356-3371. doi:10.3864/j.issn.0578-1752.2020.16.013

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【Objective】 The purposes of this study were to illuminate the effects of different packaging on energy metabolism of Flammulina filiformis from mitochondrial level, providing a new idea for further revealing the preservation mechanism of nano-package. 【Method】 In this experiment, F. filiformis was used as the research object. By using three common methods, including discontinuous density gradient centrifugation method (DDGCM), yeast mitochondrial extraction kit method (YMEKM) and improved Pulilai kit method (IPKM), the optimal way to extract F. filiformis mitochondria was determined. The regularity of post-harvest energy metabolism in packaged F. filiformis was expounded via measuring the content of ATP metabolism system substances (ATP, ADP, AMP and energy charge) and mitochondrial complex activity. 【Result】 The alcohol dehydrogenase activity in mitochondria and the cytochrome C oxidase activity in cytosol were the lowest, and mitochondrial respiration rates were the highest in IPKM extracted samples compared to other two methods, demonstrating the IPKM obtained mitochondria had the best membrane integrity. The mitochondrial superoxide dismutase activity extracted by IPKM was the highest, reaching 17.82 U·mg^-1 pro, which was 7.31% and 25.59% higher than that extracted by DDGCM-1 and YMEKM, respectively. Combined with the results of transmission electron microscopy and Janus green B staining test showed that the IPKM treatment had the largest number of active mitochondria, and their mitochondrial structure was relatively complete. During the cold storage period of F. filiformis, the sample in nano-packaging (NP) groups showed that not only the ATP and energy content but also the activity of the main mitochondrial complex was significantly higher than those of polyethylene (PE) packaged bags (control). NP materials promoted the process of oxidative phosphorylation of mitochondria by maintaining a high level of ATP content and delaying the decline of energy charge. At the 21st day, the ATP content of NP groups was still 113.83 μg·g^-1FW, which was higher than that of control, thus avoiding the reduction of the activity of F. filiformis mitochondrial complex I and Ⅲ, and slowing down the activity of mitochondrial complex Ⅳ. The mitochondrial complex Ⅳ activity of F. filiformis in the PE packaging groups reached the first peak at the 9th day, while NP groups reached the first peak at the 15th day, and the activity of the mitochondrial complex IV (5.14 U·mg^-1 pro) in the NP materials reached the first peak on the 15th day, which was significantly higher than that in the PE packaging groups (1.12 U·mg^-1 pro), with a difference of 78.23%. It proved that NP could effectively control the electron transfer in the mitochondrial respiratory chain without hindrance, ensure the energy supply of cells, thus better maintain the energy state of F. filiformis . 【Conclusion】 Compared with other methods, the mitochondria proposed by IPKM was the best approach for leading to the minimal damage on mitochondria structure and bioactivity, which laid the foundation of mitochondrial energy metabolism research on F. filiformis in the future. Meanwhile, the energy state of F. filiformis was maintained and its shelf-life was prolonged by NP treatment via improving ATP content and mitochondrial complex I, Ⅲ activities, and delaying the decline of energy charge and mitochondrial complex Ⅳ activity.

Influence of Oxidative Modification by Malondialdehyde on Structure and Emulsifying Properties of Walnut Protein

WANG YaoSong,ZHANG WeiWei,MA TianYi,CAI Min,ZHANG YiFan,HU RongRong,TANG ChangBo

Scientia Agricultura Sinica. 2020, 53(16): 3372-3384. doi:10.3864/j.issn.0578-1752.2020.16.014

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【Objective】The objective of this study was to investigate the effects of malondialdehyde (MDA), a final product of lipid peroxidation, on the structure and emulsifying properties of walnut protein isolate (WPI). Understanding the molecular mechanism, by which the oxidative modification of WPI influences its functional property, could provide a theoretical basis for mitigating oxidative deterioration of WPI-stabilized emulsion.【Method】The walnut protein was isolated by alkali dissolution and acid precipitation. MDA was generated by adding 1,1,3,3-tetraethylcyclopropane (final concentrations of 0, 0.1, 1, 5, 10 and 20 mmol·L^-1) to react with the WPI at room temperature for 24 h. Excess MDA was removed by dialysis, and MDA-oxidized WPI was freeze-dried prior to analysis. Oxidative stress markers (thiol, disulfide bond, amine and carbonyl group), physicochemical properties (solubility, turbidity, hydrophobicity, particle size, and potential), and emulsifying properties of the oxidized WPI were analyzed.【Result】Oxidation induced by MDA, particularly at 5-20 mmol·L^-1, significantly reduced the solubility of WPI from 68.74% to 11.88%, but had no noticeable effect on the turbidity of protein solutions (the values for all samples maintained both at approximate 0.32). MDA had little effect on the total thiol group, disulfide bond, free amino group and carbonyl group at 0-1 mmol·L^-1, but significantly decreased the total thiol group and free amino group and increased disulfide bond and carbonyl content at higher concentrations. SDS-PAGE results confirmed that MDA with a concentration above 5 mmol·L^-1promoted the formation of intra/inter-molecular disulfide bonds and other covalent bonds between the walnut proteins. MDA did not alter the secondary structure of WPI at below 0.1 mmol·L^-1, but significantly reduced the content of α-helix, β-sheet and β-turn and increased the content of random coil at above 1 mmol·L^-1. The decline in fluorescence intensity of WPI with the increase of MDA concentration was parallel to the disordered change in protein secondary structure, especially for MDA concentrations above 10 mmol·L^-1. Oxidation by <1 mmol·L^-1 MDA had no remarkable effect on the reduction, but >1 mmol·L^-1 MDA significantly reduced protein hydrophobicity to as little as 1/10 of the control. MDA at less 1 mmol·L^-1 had no impact on the protein particle sizes and their change, whereas MDA at above 1 mmol·L^-1 significantly increased protein particle size and reached a maximum particle size of approximate 1 160 nm at 10 mmol·L^-1 MDA. Meanwhile, the oxidation significantly reduced the charge of the protein with the increase of MDA concentration. Oxidation by 0.1 mmol·L^-1 MDA significantly reduced protein emulsifying activity, while oxidation by 1 mmol·L^-1 MDA significantly reduced emulsifying stability. The maximum loss of 2/3 of the total emulsifying index was observed when the MDA concentration reached 20 mmol·L^-1. 【Conclusion】As the degree of oxidation increased in the oxidation system, MDA as byproducts derived from lipid peroxidation, significantly modified the protein structure (including residue groups), promoted protein cross-linking and formation of large aggregates, and changed the physical and chemical properties of WPI. This resulted in a significant loss of the emulsifying properties of WPI.

Effects of Saccharomyces Cerevisiae and Bacillus Licheniformis on Growth Performance and Rumen Fermentation in Sheep

ZHENG WeiCai,HAO XiaoYan,ZHANG HongXiang,XIANG BinWei,ZHANG WenJia,ZHANG ChunXiang,ZHANG JianXin

Scientia Agricultura Sinica. 2020, 53(16): 3385-3393. doi:10.3864/j.issn.0578-1752.2020.16.015

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【Objective】The objective of this experiment was to study the effects of Saccharomyces cerevisiae and Bacillus licheniformis on growth performance and rumen fermentation in sheep. 【Method】A total of 48 four-month-old healthy Dorper×Small Tail Han sheep ram lambs with similar body weight (22.96±2.00 kg) were randomly selected into 4 groups: D (the control group, without both bacteria); D1 (only adding Saccharomyces cerevisiae, 6×10¹⁰CFU/kg), D2 (only adding Bacillus licheniformis, 2×10¹⁰CFU/kg), and D3 (Saccharomyces cerevisiae 6×10¹⁰CFU/kg + Bacillus licheniformis 2×10¹⁰CFU/kg), with 12 sheep of each group. The experiment was conducted over 75d, with a 15d adaptation and a 60d the formal period. The experimental sheep were fed 2 times per day (08:00 a.m., 18:00 p.m.), free ingestion and free drinking water. During the normal feeding period, each experimental sheep were recorded accurately the amount of feed and residual of weight every day. The experimental sheep were weighed before the morning feeding on the 1st, 30th and 60th day, and the average daily intake, average daily gain (ADG) and Feed/Gain (F/G) value were calculated. At 08:00 on the day of the end of the experiment, the experimental sheep were fed normally. After 3 hours, the rumen fluid was collected and the rumen fermentation, digestive enzyme activities, and functional microorganisms were determined. 【Result】The results showed as follows: (1) the addition of Saccharomyces cerevisiae and Bacillus licheniformis in the diet had no significant effect on the final body weight and average feed intake of the test sheep (P>0.05). The ADG of D3 group was significantly higher than that of D group (P<0.05), and F/G value of D3 group was significantly lower than that of D group (P<0.05). There was no significant difference between D1 and D2 groups in F/G value (P>0.05): (2) the addition of Saccharomyces cerevisiae and Bacillus licheniformis in the diet had no significant effect on the rumen pH value, the concentration of butyric acid, and the value of Acetate/Propionate (A/P). The concentration of NH₃-N for D3 group was significantly lower than D group, but the concentration of TVFA and propionic acid was significantly higher than D group (P<0.05), with no difference among three experimental groups (P>0.05). The concentration of acetic acid in D3 and D2 group was significantly higher than that in D1 and D groups (P<0.05).3)The activity of β-glucosidase, carboxymethyl cellulose, xylanase, and amylase for D3 group was significantly higher than the other groups (P<0.05). There was no significant effect in the activities of β - glucosidase, pectase, carboxymethyl cellulose and amylase among D , D1 and D2 groups (P>0.05). The activity of protease in D3 group was significantly higher than D and D2 groups (P<0.05), with no significant difference between D3 and D1 groups(P>0.05). There was no significant difference in β-glucosidase, pectase, carboxymathy cellulose, xylanase, protease and amylase activities between D1 and D2 groups (P>0.05); (4) the addition of Saccharomyces cerevisiae and Bacillus licheniformis in the diet had no significant effect on the amount of R. flavefaciens, P. ruminicola, R. amylophilus, and Protozoan(P>0.05). The amount of B.fibrisolvens for D3 group was significantly higher than the other 3 groups (P<0.05). The amount of R. albus and F. succinogensin D3 group were significantly higher than D group (P<0.05), with no significant difference among D1, D2 and D3 groups (P>0.05). Compared with the D group, the amount of Methanogensin experimental groups were significantly decreased (P<0.05), and the D3 group was lowest. 【Conclusion】In conclusion, the dietary supplementation of Saccharomyces cerevisiae (6×10¹⁰CFU/kg) and Bacillus licheniformis (2×10¹⁰CFU/kg) had positive effects on rumen fermentation, which improved rumen digestive enzyme activity and increased the number of beneficial bacteria in rumen. Moreover, the combination of Saccharomyces cerevisiae and Bacillus licheniformis achieved better effect.

Construction of lpxM Gene Deletion Strain of Haemophilus parasuis and It's Some Biological Characteristics

YANG Jun,CHU PinPin,SONG Shuai,CAI RuJian,YANG DongXia,BIAN ZhiBiao,GOU HongChao,LI Yan,JIANG ZhiYong,LI ChunLing,YAN He

Scientia Agricultura Sinica. 2020, 53(16): 3394-3403. doi:10.3864/j.issn.0578-1752.2020.16.016

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【Objective】The effects of lpxM, a gene related to lipopolysaccharide synthesis of Haemophilus parasuis, on its some biological characteristics such as growth, biofilm formation ability, antibacterial ability against 50% porcine serum, virulence to macrophage and antibiotic susceptibility were studied, which laid a certain theory for revealing the pathogenesis of HPS and the construction of lpxM gene deletion vaccine, and also provided a basis for the selection of drugs for the prevention and treatment of HPS on pig farms.【Method】The highly pathogenic serotype 5 of HPS local isolate H45 was used as the research object, and the suicide plasmid PK18mobsacB was used as a vector. The constructed recombinant plasmid was transformed into H45 by natural transformation method, and homologous recombination occurred under the pressure of antibiotics. Finally, the lpxM gene deleted strain H45-ΔlpxM was obtained by antibiotic screening, and verified by PCR and sequencing. The differences in biological characteristics between the two were compared. The OD600-t curve of the two was used to compare the growth conditions. The ability of the two to form biofilm formation after 24 hours of culture was compared by crystal violet staining. The survival rate of the two in 50% porcine serum was compared, and the antiserum complements of the two were compared. The two both simultaneously stimulated mouse monocyte macrophage RAW264.7 cells for 6, 12 and 24h, and detected the release of LDH in cell culture supernatant for comparing the virulence of macrophages. The difference in antibiotic susceptibility was studied by KB diffusion method. Antibiotics included 13 antibiotics in clinical practice, such as ampicillin and polymyxin B antibiotics. The sensitivity to antibiotics was determined by measuring the diameter of the inhibition zone according to the resistance standard. 【Result】 The lpxM gene deletion strain H45-ΔlpxM was successfully constructed. The growth of the deleted strain was found to be slower than the wild-type strain in the early growth stage, but they were consistent after 8 hours. The results showed that the lpxM gene deletion could inhibit the growth of H45 to some extent. The two both could form biofilm, but the deleted strain was weaker than the wild strain. The wild strain had a survival rate of 16.1% in 50% porcine serum, while the deletion strain was only 0.71%, and the deletion strain was significantly lower than the wild strain. The two both could cause macrophage death. The lethal rate of wild-type plants was 6.63%, 10.86% and 22.17%, after 6, 12 and 24h, respectively, while the lethal rate of the deleted strain was 2.62%, 6.35% and 18.01%, respectively. With the prolongation of action time, the virulence effect was more obvious, and had a certain time dependence. At each time point, the virulence of the deleted strains was lower than that of the wild strain. The antibiotic sensitivity results showed sensitive to ten antibiotics such as thiophene and showed resistance to enrofloxacin. But the resistance to amoxicillin clavulanic acid, sulfamethoxazole and ampicillin changed greatly. Amoxicillin clavulanic acid and sulfamethoxazole became sensitive from resistance and ampicillin also changed from intermediate to sensitive. The results showed that lpxM gene deletion had certain influence on some antibiotic sensitivity of H45.【Conclusion】 The deletion of lpxM gene could inhibit growth of HPS to a certain extent, reduce its biofilm formation ability, anti-serum bactericidal ability and virulence to macrophages, and increase sensitivity to some commonly used antibiotics, revealing that lpxM gene might be the virulence gene of HPS and be closely related to the pathogenic ability of HPS, but the specific mechanism needed further study.

Construction and Utilization of Database for Chinese Maize Varieties and Their Genealogy

LI JianXin,XI MengHui,ZHANG JiaWei,XI MengJuan,TIAN Ding,LU YiZhe,CHEN XiaoYang,LI WeiHua,ZHANG XueHai,TANG JiHua

Scientia Agricultura Sinica. 2020, 53(16): 3404-3411. doi:10.3864/j.issn.0578-1752.2020.16.017

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【Objective】 The breeding and popularization of new varieties are important for promoting the continuous improvement of maize yield in China. Breeding elite inbred lines is the prerequisite for strong heterotic combinations. To determine the kinship and genealogy relationship among maize inbred lines is the important basis for improvement of maize inbred, classification of heterotic groups and cross combination. It is imperative to sort out the genealogy of existing varieties and construct the corresponding genealogy database, so as to solve unclear consanguinity and inconvenient inquiry of maize inbred lines in China. 【Method】 A network MySQL database (https://www.maizedata.cn) of Chinese maize inbred lines, varieties and their genealogies was built based on the LNMP (Linux system, Nginx server software, MySQL database management system and PHP hypertext preprocessing software) server environment configuration. In addition, information of inbred lines and approval information of maize varieties in past years from the websites of Ministry of Agriculture and Rural Affairs of the People's Republic of China, Provincial Agricultural and Rural Affairs Departments, the big data platform of China’s seeds, as well as related literature and books were collected, collated and imported into MySQL database. Then, dream-weaving content management system (DedeCMS) was used to manage and update MySQL database. Query requests were submitted through AJAX (Asynchronous Javascript and XML) asynchronous processing, and the extracted inbred line and variety information were processed by PHP. Finally, the detailed genealogy information and genealogy tree were returned and displayed on the front-end web pages. In addition, phpMyAdmin software was applied to optimize the database structure and add new functions. 【Result】 In this database, more than 10 000 maize varieties that examined and approved by the provincial level or above, and their parents were collected, including 1218 inbred lines and 7823 varieties that have 1 to 10 pieces of information with different approved numbers and across eras in China. Variety information retrieval, genealogy tracing, filial generation query and customized query are the core functions of the database. Additionally, functions, such as addition, modification and deletion of maize inbred lines and variety pedigree information, haven been also integrated in the database. 【Conclusion】 Based on the LNMP server environment configuration, a database for Chinese maize varieties and their genealogies was constructed, which covers more than 10 000 maize varieties, including 1218 inbred lines and 7823 varieties. Core functions contain variety information retrieval, genealogy tracing, filial generation query, customized query, data uploading and updating. As an effective tool for Chinese maize breeders and researchers to quickly search the information and pedigree of maize inbred and varieties, the database can promote the breeding of elite maize inbred lines.

Multi-Gene-Based PCR Detection and Identification of Chilli veinal mottle virus

YANG HongKai,YANG JingWen,SHEN JianGuo,CAI Wei,GAO FangLuan

Scientia Agricultura Sinica. 2020, 53(16): 3412-3420. doi:10.3864/j.issn.0578-1752.2020.16.018

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【Objective】Chilli veinal mottle virus (ChiVMV), one of the most destructive pathogens causing server losses to chilli production, is an important plant virus in port quarantine. The objective of this study is to establish a fast and accurate multi-gene-based PCR detection method for ChiVMV.【Method】DAS-ELISA and RT-PCR were used to detect the infected chilli samples imported from India. Two pairs of specific primers were designed from the conserved regions of ChiVMV coat protein (CP) and cytoplasmic inclusion protein (CI), respectively. The multi-gene-based PCR detection method was established after optimizing parameters including primer dosage and annealing temperature. The established multi-gene-based PCR detection system was also used to detect chilli viruses including ChiVMV to verify the specificity of this system, and the different concentrations of cDNA in ChiVMV positive samples were amplified to determine its sensitivity. In addition, the utility of the system was tested by detecting ChiVMV in plant samples infected by this virus.【Result】All samples reacted positive on the DAS-ELISA test. RT-PCR amplifications of the ELISA-positive subsamples all generated expected fragments of 861 bp in size, using the specific primer pair of CP861-F/CP861-R. These results indicated that all samples were infected by ChiVMV. The specific target fragments of 337 and 655 bp were respectively amplified using the primer pairs of CP337-F/CP337-R and CI655-F/CI655-R in the multi-gene-based PCR detection system, whose optimized reaction system is cDNA 2 μL, CP337-F/CP337-R 0.625 μL (10 μmol·L^-1), CI655-F/CI655-R 1.375 μL (10 μmol·L^-1), 2×PCR Master Mix 12.5 μL, ddH₂O 6.5 μL, annealing temperature 50℃, and for 35 cycles. The established multi-gene-based PCR detection system had good specificity and sensitivity, and two targeted fragments could be detected after the total DNA was diluted to 10^-4. Other ChiVMV-infected samples were successfully detected by this method, generating two expected fragments of CP and CI, respectively.【Conclusion】The established multi-gene-based PCR detection method has strong specificity, high sensitivity, excellent repeatability, which is useful in the detection and identification of ChiVMV in port quarantine.

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