Table of Content

01 January 2020, Volume 53 Issue 1

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Explore Regulatory Genes Related to Maize Leaf Morphogenesis Using RNA-Seq

ShuLei GUO,XiaoMin LU,JianShuang QI,LiangMing WEI,Xin ZHANG,XiaoHua HAN,RunQing YUE,ZhenHua WANG,ShuangGui TIE,YanHui CHEN

Scientia Agricultura Sinica. 2020, 53(1):  1-17.  doi:10.3864/j.issn.0578-1752.2020.01.001

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【Objective】Leaf shape characteristics are one of important agronomic traits that determine plant morphology and affect planting density. However, the molecular mechanism related to leaf shape remain unknown in maize. Here, transcriptome sequencing technology was used to screen and explore genome-wide analysis of regulatory genes and metabolic pathways involved in leaf morphogenesis. This study will lay the foundation for further understanding the regulator mechanism of leaf development in plant and identifying candidate genes of leaf shapes, such as leaf width and leaf length.【Method】 Extreme narrow-leaf inbred line NL409 and wide-leaf line WB665 were selected as the experimental materials. By RNA-Seq technology, the differentially expressed genes (DEGs) of the seventh leaf base between these two lines were identified during the 7th leaf stage. Furthermore, metabolic pathways closely related to leaf development were also analyzed using a series of bioinformatics analysis. qRT-PCR was used to validate the expression level of DEGs in different hormone pathways, and the further promoter analysis were performed to explore leaf-shape functional genes.【Result】By analyzing the high-throughput sequencing in WB665 and NL409, a total of 5 199 DEGs were obtained at the primary section of leaf width formation. Of which, 2 264 (43.55%) genes were up-regulated, whereas down-regulated genes were significantly more than up-regulated genes with 2 935 (56.45%) decreased genes. GO enrichment analysis showed that these DEGs were mainly enriched in cell membrane-associated function terms of cellular components, including metabolic process and cell stimulus response. KEGG enrichment analysis showed that these DEGs were mainly involved in ribosome, plant hormone signal transduction, sphingolipid metabolism pathways, phenylpropanoid biosynthesis, glyoxylate and dicarboxylate metabolism and other processes. among which ribosome, plant hormone signal transduction, sphingolipid metabolism pathways with more down-regulated genes were closely related to leaf development. One of PRS (PRESSED FLOWER) family genes, which were enriched in the ribosomal pathway in this study, PRS13 (PFL2) was identified to participate in regulating the development of narrow leaves. The expression pattern of genes enriched in sphingolipid metabolism pathway and its related MAP kinase, AP1-like, and LFY-like were consistent with the result of the inhibited development of narrow leaves. Notably, all of BR (Brassinosteroid) response genes and most of GA (Gibberellin) metabolic genes were down-regulated in plant hormone signal transduction pathway, while the expression level of all the CTK (Cytokinine) response genes and Auxin genes are mostly increased. The action of up-regulated expression of DELLA protein gene affecting the GA and CTK pathways was consistent with the phenotypic result of narrow leaves. Eighteen genes were validated by qRT-PCR. The result showed that the expression trend was consistent with the transcriptome data. Moreover, the BR-related ROT3, auxin-related NAL7-like, AGO7-like and TCP-like transcription factors CYC/TB1 were identified to be closely associated with the formation of narrow leaves.【Conclusion】Summarily, this study unveils several metabolic pathways closely related to leaf development in maize, and find the dynamic balance between plant hormones plays an important role in leaf development, especially the interaction between Auxin and BR as well as CTK and GA.

The Mechanism of Ectopic Expression of Brassica juncea Multidrug and Toxic Compound Extrusion (BjMATE) to Enhance the Resistance to Acid and Aluminum Stress in Alfalfa

XiaoDong LI,YiShun SHANG,ShiGe LI,GuangJi CHEN,ChengJiang PEI,Fang SUN,XianQin XIONG

Scientia Agricultura Sinica. 2020, 53(1):  18-28.  doi:10.3864/j.issn.0578-1752.2020.01.002

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【Objective】 Acid and aluminum stress is one of the major environmental stresses in agricultural production in the south of China. Studying the mechanism of native plants adaptation to acid and aluminum stress, and identifying and making use of the excellent gene resources would benefit to breed new varieties of acid and aluminum resistant crops, that will play a fundamental role in molecular breeding of alfalfa. 【Method】 In previous study, full length of BjMATE was cloned from Pingba bitter rape, a native Brassica juncea resource in Guizhou province, and the overexpression vector was constructed by the alfalfa genetic improvement group of Guizhou Institute of Prataculture. The transgenic plants of alfalfa and Arabidopsis thaliana were obtained by tissue culture and flower dipping methods, respectively. Molecular examination was carried out with NPT resistance gene primers and gene-specific primers; and the expression of BjMATE in transgenic plants was examined by qRT-PCR. The germination rate and seedling growth of transgenic and control alfalfa lines were analyzed by hydroponic method under acid, aluminum and acid aluminum combination stress conditions. The alteration of the seedling and root growth was also analyzed between transgenic and Zhongmu 1# alfalfa under a long term but weaker acid-aluminum stress condition. Antioxidant enzyme activity, including POD, SOD, CAT, and MDA concentration alterations between OEMs-5 and Medicago sativa Zhongmu 1# were analyzed with microplate reader under acid, aluminum and acid-aluminum combined stress conditions. At the same time, expression changes of AtMATE, AtPIN2, AtALS3, AtALMT1, and AtSTOP1 which were key genes involving in acid aluminum regulation pathways, were analyzed in the model plant Arabidopsis thaliana by qRT-PCR, and potential regulation network were discussed. 【Result】 The positive rates of transgenic Arabidopsis and alfalfa were 100% and 66.7%, respectively. Compared with the control lines, the expression levels of the candidate transgenic alfalfa and Arabidopsis lines were up-regulated by 63.02 and 76.87 times, respectively. There was no significant difference in seed germination rate between OEMs-5 and Zhongmu 1# under normal, acid stress, aluminum ion stress and acid-aluminum combination stress conditions. While the germination potential was significantly different among the treatments, as well as the materials, OEMs-5 was significantly better than Zhongmu 1#. After a long-term weak stress treatment, the plant height of OEMs-5 was not significantly different from that of Zhongmu 1#; but the biomass and root length of OEMs-5 was significantly better than that of Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Antioxidant enzyme activity and MDA concentration alterations between OEMs-5 and Zhongmu 1# were different. POD and SOD activity increased in both OEMs-5 and Zhongmu 1# after stress treatment, however, significant difference was only detected in SOD assay. No significant difference in CAT activity was detected between the two materials, and the treatments. The content of MDA decreased slightly, significant differences were detected between OEMs-5 and Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Expressions of AtMATE, AtPIN2, AtALS3, AtALMT1 and AtSTOP1 were up-regulated under aluminum stress and acid-aluminum combined stress in both OEMs-5 and Zhongmu 1#, however, no significant difference were detected between them except AtSTOP1.【Conclusion】BjMATE can positively regulate alfalfa tolerance to acid-aluminum stress during seed germination and seedling growth stages. Reactive oxygen species (ROS) scavenger system, especially the activity of SOD, and AtSTOP1 gene which is a key gene involving in acid-aluminum stress regulation pathway, may participate in BjMATE mediated acid-aluminum stress regulation.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Straw Returning Amount on Grain Yield, Dry Matter Accumulation and Transfer in Summer Maize with Different Genotypes

XinYuan MU,Xia ZHAO,LiMin GU,BaoYi JI,Yong DING,FengQi ZHANG,Jun ZHANG,JianShuang QI,ZhiYan MA,LaiKun XIA,BaoJun TANG

Scientia Agricultura Sinica. 2020, 53(1):  29-41.  doi:10.3864/j.issn.0578-1752.2020.01.003

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【Objective】 Scientific and objective evaluation of the comprehensive effect of straw returning is important to improve the utilization rate of straw resources and to promote the green and sustainable development of agriculture. In this study, a field located experiment was conducted in wheat-maize cropping system on the North China Plain, to clarify the responses of different genotypes summer maize to various straw returning amounts. The purpose of this paper was to provide support for the comprehensive evaluation of the straw returning technology and for the optimization of straw returning amount. 【Method】 The experiment was conducted from October 2017 to October 2018 at Yuanyang, Henan province. The experimental design was a split plot. The main plot was four straw returning amount, no straw returning to field (S0), 50% amount of straw returning to field (S1), 100% amount of straw returning to field (S2) and 200% amount of straw returning to field (S3), and the subplot was genotype, including XD20 and ZD958. The differences in leaf area index (LAI), dry matter accumulation and transfer, and grain yield under different treatments were analyzed. 【Result】 Compared with no straw returning to field, the straw returning increased LAI after silking, and there was a significant interaction between genotypes and straw returning amount on LAI. At anthesis stage, the LAI of XD20 and ZD958 increased with the increasing of straw returning amount, and were the highest under S3 treatment. At maturity stage, with the increasing of straw returning amount, the decline in LAI for XD20 and ZD958 showed a trend of first decreasing and then increasing, and was the lowest under S1 for XD20 and S2 for ZD958. In addition, the LAI of XD20 and ZD958 at maturity stage showed a trend of first rising and then falling with the increasing of straw returning amount, and were the highest under S1 for XD20 and S2 for ZD958. Higher post-silking LAI was conducive to maintain higher post-silking dry matter production capacity, eventually significantly increased post-silking dry matter accumulation and optimize dry matter accumulation and distribution characteristics. The interaction of genotype and straw returning amount significantly affected the dry matter remobilization (DMR), dry matter remobilization efficiency (DMRE), contribution of dry matter remobilization to grain (DMRCG), dry matter accumulation after anthesis (DMAA) and contribution of dry matter accumulation after anthesis to grain (DMAACG). With the increasing of straw returning amount, DMR, DMRE and DMRCG of the vegetative organs (stem sheath + leaf) in XD20 firstly decreased and then increased, and were the lowest under S1 treatment. Meanwhile, DMAA and DMAACG showed an increasing trend first and then a decreasing trend, which were the highest under S1 treatment. The DMR, DMRE, DMRCG and DMAA of the vegetative organs (stem sheath + leaf) of ZD958 all showed an ascending and then descending trend with increasing of straw returning amount, and those were the highest under S2 treatment. The increase of dry matter accumulation was beneficial to increase grain weight and grain yield. Compared with S0 treatment, S1, S2 and S3 treatment improved the grain yield, but the grain yield did not increase continuously with the increasing of straw returning amount. For XD20, the highest grain yield was obtained under S1 treatment, which was 3.5% to 17.7% averagely higher than other treatments. For ZD958, the highest grain yield was achieved under S2 treatment, which was 0.4% to 16.8% averagely higher than other treatments. 【Conclusion】 Our results showed that, for wheat-maize cropping system on the North China Plain, appropriate amount of straw to the field could delay the leaf senescence process in the later growth stage of maize, optimize the dry matter accumulation and distribution characteristics, improve the post-silking dry matter accumulation and grain weight, and thereby improve the grain yield of maize. However, the responses of different maize cultivars to straw returning amount vary greatly. When promoting straw returning to the field, the effects of crop genetic factors on straw returning should also be considered.

High-Throughput Phenotyping of Individual Wheat Stem and Ear Traits with Machine Vision

QiShuo DING,HaiKang LI,KeRun SUN,RuiYin HE,XiaoChan WANG,FuXi LIU,Xiang LI

Scientia Agricultura Sinica. 2020, 53(1):  42-54.  doi:10.3864/j.issn.0578-1752.2020.01.004

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【Objective】 High-throughput phenotyping (HTP) is not only an important tool of modern agriculture for crop breeding, but also a powerful means to illustrate physiological and ecological mechanisms of crops in the field. However, the basic features of structural components of each HTP tools have to be illustrated. It is therefore necessary to investigate what a technical feature is applicable to machine vision based HTP system.【Method】 An image-processing tool was developed to measure stem-and-ear level traits of each individual wheat stem. Three wheat species, i.e. Ningmai 13, Luyuan 502 and Zhengmai 9023, were used for plot experiment analysis. The wheat was sown with luffer board having equally spaced seeding holes. The precision seeding tools were applied to control wheat population accurately. At the maturity of post-paddy wheat, the integrated image of stem and ear was obtained, and the image was subjected to gray enhancement, histogram equalization, S component extraction, Otsu threshold segmentation, stem and ear separation, and stem and ear morphology parameters. The morphological parameters of the individual organs per stem-panicle of the extracted post-paddy wheat included stem length, average stem width, stem projection area, stem circumference, ear length, average ear width, ear projection area and ear circumference. In addition, traditional methods of measurement were used to derive single leaf weight, single stem weight, single ear weight and single ear yield etc. Linear, quadratic, extended and exponential models were applied for the regression on the collected multi-dimensional data sets, including correlations between ear and stem level biomass and individual ear grain yield, interrelationships among morphological parameters of stem and ear and single ear grain yield. Correlation analysis and regression analysis were performed on the processed indices of wheat. Based on this case study, some key aspects of technologies were discussed concerning on the application of machine vision tools on high-throughput phenotyping in the field.【Result】Results showed that correlation coefficients of individual stem and leaf weight with individual ear grain yield decreased steadily from Ningmai 13 to Luyuan 502, and till Zhengmai 9023. Correlation coefficient of stem and ear morphological parameters with individual ear grain yield was significantly lower than that among the biomasses. However, composite morphological parameter, which integrated single ear projection area and single ear length, was found significantly correlated with individual ear grain yield. The best regression model for the correlation between stem and ear biomass and individual ear grain yield of the three wheat species were different. Morphological parameters derived from ear images failed to predict individual ear grain yield precisely. However, combined morphological parameters from wheat stem and wheat ear revealed the best result of regression with extension models. Composite morphological stem-and-ear level traits of individual wheat stem provided more accurate prediction on the ear-derived grain yield, which could make the yield prediction with growth-stage traits collected with machine vision technically possible. Machine vision tools of HTP provided a much higher sets of agronomic trait indices as compared with traditional methods, providing more options for the illustration on the correlations among agronomic traits and path-analysis on crop yield. It in turn resulted into high-dimensional data sets and technical difficulties impeding the identification on valuable information. 【Conclusion】A basic infrastructure of HTP machine vision tools for field wheat stand was defined as multi-scale and automatic adaptation aspect. It should be autonomously adaptable to multi-scales concerning with the field, crop stand, individual crop and organ-level traits of each individual crop. It also provided traits identification and calculation with statistical analysis on different physiological periods of wheat, e.g. seedling stage, tillering stage, jointing stage etc. Meanwhile, in each development stage of the machine-vision HTP tools and for each functional module, in-depth involvement of agronomical calibration was required. In safeguarding the reliability of machine-vision tools, standardization on referencing HTP-derived traits was also necessary.

PLANT PROTECTION

FliZ Regulated the Biofilm Formation of Bacillus subtilis Bs916 and Its Biocontrol Efficacy on Rice Sheath Blight

HuaFei ZHOU,HongFu YANG,KeBing YAO,YiQing ZHUANG,ZhaoLin SHU,ZhiYi CHEN

Scientia Agricultura Sinica. 2020, 53(1):  55-64.  doi:10.3864/j.issn.0578-1752.2020.01.005

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【Objective】The objective of this study is to discover and identify new regulatory genes on biofilm formation of Bacillus subtilis Bs916, detect its effect on biofilm formation of Bs916 and biocontrol efficacy on rice sheath blight.【Method】The single knockout mutant of Bs916 at fliZ was construct by homologous recombination, and its defects in biofilm formation were verified by dry weight analysis. The anti-bacterial effect of fliZ mutant and Bs916 on rice sheath blight pathogen (Rhizoctonia solani) was detected by flat panel. The relative production of 3 lipopeptide antibiotics (LPs) surfactin, bacillomycin L, and fengycin in fliZ mutant and Bs916 was detected by HPLC. The GFP-labeled strains of Bs916 and fliZ mutant were constructed by green fluorescent labeling, the colonization ability of them in rice stalks was observed, and the biocontrol efficacy of fliZ mutant and Bs916 on rice sheath blight was detected.【Result】The single knockout mutant of Bs916 at fliZ was successfully constructed. Compared with the three-dimensional structure biofilm of the control group Bs916, fliZ mutant only formed a planar two-dimensional structure biofilm, appeared broken form, which proved that it had significant defects in biofilm formation. Quantitative analysis of the dry weight of biofilms showed that the biofilm dry weight of fliZ mutant was only 23% of the control group Bs916, which further verified that the biofilm formation ability of fliZ mutant was significantly decreased. The motility test found that the expanded diameter of fliZ mutant was only 32% of Bs916, which proved that the swimming ability of fliZ mutant was significantly reduced. The bacteriostatic test showed that the antibacterial bandwidth of the two strains was basically the same, and it is proved that the antibacterial activity of fliZ mutant against R. solani was not significantly different from that of Bs916. The relative production of three LPs bacillomycin L, surfactin, and fengycin in fliZ mutant and Bs916 was successfully detected. Compared with Bs916, the relative production of bacillomycin L was significantly increased by 1 time in fliZ mutant, but the relative production of surfactin and fengycin was not significantly different from that of Bs916. The colonization test of rice stalks showed that the number of fliZ mutant was significantly lower than that of Bs916, and there was no significant aggregation effect near the rice sheath blight lesions, and presented an unordered state, which proved that the colonization ability of fliZ mutant on rice stalks was significantly lower than that of Bs916. The field biocontrol trials against rice sheath blight showed that biocontrol efficacy of fliZ mutant ranged from 6.0% to 20.7% on days 6-15, which was significantly lower than that of Bs916 (36.0%-57.6%). It was proved that the biocontrol efficacy of fliZ mutant on rice sheath blight was significantly reduced.【Conclusion】The new regulatory gene fliZ of Bs916 biofilm identified in this study is located in the signal pathway controlling flagellar movement, directly acts on swimming and expansion of the bacteria, and can significantly control the biofilm formation and its biocontrol efficacy on rice sheath blight.

Functional Analysis of Gene ShARPC5 Involved in Tomato Resistance to Powdery Mildew

ChanJing FENG,GuangZheng SUN,Yang WANG,Qing MA

Scientia Agricultura Sinica. 2020, 53(1):  65-73.  doi:10.3864/j.issn.0578-1752.2020.01.006

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【Objective】Powdery mildew is an important disease in tomato production, and seriously affects the yield of tomato. Genome sequences of Solanum lycopersicum provide valuable information resources for disease-resistant gene searching. The actin- related protein 2 and 3 complex (ARP2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. In this study, tomato ARPC5, encoding a subunit protein of the ARP2/3 complex, was cloned and identified for disease resistance. The results will lay a foundation for upgrading tomato genomic information, further studying the resistance mechanism and molecular breeding.【Method】ShARPC5 was annotated from the genomic databases and cloned from tomato LA1777 (S. habrochaites). DNAMAN 6.0 was used for multiple sequence alignment and MEGA 6.0 was used for constructing phylogenetic tree. The prediction of protein subcellular localization was performed using ProtComp v. 9.0. The qRT-PCR was used to compare the expression of ARPC5 in the high-sensitive variety Moneymaker (MM) and high-resistant variety LA1777 induced by Oidium neolycopersici (On-Lz). The correlation between On-Lz infection and ARPC5 expression was analyzed. The function of ShARPC5 involving in tomato resistance to On-Lz was further verified by the technology of virus-induced gene silencing (VIGS). The phenotypic changes of ShARPC5-silenced and wild-type lines were observed. Hypersensitive response (HR) and H₂O₂ production were detected by trypan blue and DAB staining, respectively, and the expression of some marker genes related to plant disease resistance was assayed in ShARPC5-silenced plants. Additionally, genetic transformation of Arabidopsis thaliana was conducted by Agrobacterium-mediated floral-dip method. The phenotypic changes of transgenic and wild-type lines were observed, and the number of conidia per lesion was also counted.【Result】The S. habrochaites ARPC5 was identified and characterized. ShARPC5 encodes a 132-amino-acid protein possessing a conserved P16-Arc domain. Compared with the compatible interaction between tomato MM (Moneymaker) and On-Lz, the expression of ShARPC5 was significantly up-regulated in incompatible interaction between tomato LA1777 and On-Lz, especially at 18 hpi. Silencing of ShARPC5 in tomato could increase the susceptibility to the powdery mildew pathogen On-Lz. The expression of PR1b1, a maker gene related to signal regulation, was significantly down-regulated after silencing of ShARPC5. The histological observation showed that the induction of hypersensitive cell death and the generation of reactive oxygen were reduced in silenced-ShARPC5 tomato plants compared with wild types. Transient over-expression of ShARPC5 in tobacco could rapidly produce necrotic spots. Conversely, over-expression of ShARPC5 in A. thaliana, followed by inoculation with On-Lz, showed enhanced resistance.【Conclusion】ShARPC5 is an important gene which can reduce the incidence of tomato powdery mildew, and has a great application value in the mechanism study of tomato resistance to powdery mildew. At the same time, it can be used as a candidate gene for molecular breeding of tomato powdery mildew resistance.

The Role of High-Level Biosafety Laboratories in Biosafety and Consideration About Their Development

Jing LIU,Chao LI,JinXiong LIU,Rui HE,YanRong SUN

Scientia Agricultura Sinica. 2020, 53(1):  74-80.  doi:10.3864/j.issn.0578-1752.2020.01.007

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Biosafety laboratory is a significant location for conducting biosafety related researches such as infectious disease prevention and control, biological invasion, and so on. At the same time, it also provides an important safety guarantee for the laboratory staff to avoid pathogen infection and prevent pathogen leakage. The biosafety laboratories are classified into four levels (including levels one, two, three, and four) of protection based on the differences of the pathogenicity, toxicity level, scope of source of infection, infectiousness, transportability, stability, and induced hypersensitivity of biomasses. The biosafety laboratories in levels three and four are also called high-level biosafety laboratories. The high-level biosafety laboratory can be used to conduct researches related to highly pathogenic and highly infectious pathogenic microorganisms, to develop rapid diagnosis and monitoring technologies, to study prevention and control techniques target to unexpected acute infectious disease, and to engage reserve researches for prevention and management of potential epidemic infection disease. Moreover, the high-level biosafety laboratory can be evolved in setting up courses for training specialized personnel. In addition, the mobile high-level (level three) biosafety laboratory can also be used for dealing with emergency biosafety threats. Furthermore, the high-level biosafety laboratory is playing a strategic role in America and Europe in combatting unexpected emergent public health events and military exploration, including “Big Health” plan, researches on Biological Dual-Use Technology, training on professional ability of emergency treatment to epidemic infectious disease. Nowadays, with the high risk of introduction of infectious diseases and complex international context, the high-level biosafety laboratory, as the infrastructure and important platform of scientific research, has been playing an important role in prevention and management of fulminating infectious diseases. However, the building, biosafety management, and equipment research and development of the high-level biosafety laboratory need to be further strengthened in China. In the present study, the building and operational conditions of the high-level biosafety laboratory in China were introduced. The roles of high-level biosafety laboratory in biosafety of national and international were discussed under the background of high incidence of new-and-outbreaks of infectious diseases. Furthermore, the problems discovered in current operation as well as the development strategies in the future were considered.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Yield Variation of Winter Wheat and Its Relationship to Yield Components, NPK Uptake and Utilization of Leading and High Yielding Wheat Cultivars in Main Wheat Production Regions of China

Ning HUANG,ZhaoHui WANG,Li WANG,QingXia MA,YueYue ZHANG,XinXin ZHANG,Rui WANG

Scientia Agricultura Sinica. 2020, 53(1):  81-93.  doi:10.3864/j.issn.0578-1752.2020.01.008

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【Objective】The aim of this study was to clarify wheat yield variation and its relationship to yield components and nutrient uptake and utilization for major high-yielding cultivars, so as to provide guidance to close yield gap and realize high yield and high quality in wheat production. 【Method】 Field experiments were conducted in 2016-2017 to test the major wheat cultivars in North-Huanghuai, South-Huanghuai, and the middle and lower Yangtze River reaches of China, under local suitable agricultural cultivations. The variation of yield and its relationship to dry matter accumulation, yield components, NPK uptake and utilization were investigated for the high-yielding cultivars in the three wheat production regions. 【Result】 Large variation of grain yield existed in each wheat production region, ranging from 7 751 to 8 702 kg hm ^-2 in North-Huanghuai, 7 302 to 8 413 kg·hm ^-2 in South-Huanghuai, and 5 554 to 6 294 kg·hm ^-2 in the middle and lower Yangtze River reaches. The high-yielding cultivars in North-Huanghuai were found to have higher biomass, harvest index and spike number than that of control cultivars, the high-yielding cultivars in South-HuangHuai had higher harvest index and grain number, and the high-yielding cultivars in the middle and lower reaches of the Yangtze River had higher harvest index and thousand grain weight. Besides, in North-HuangHuai the high-yielding cultivars showed lower grain N content and N requirement, and higher N physiological efficiency than that of control, in South-HuangHuai the high-yielding cultivars showed lower straw P content and P requirement, higher P physiological efficiency and straw K content, and in the middle and lower Yangtze River reaches, the high-yielding cultivars showed lower K content in grain, higher K content in straw, P content in grain and straw, and N and P uptake in shoot, and lower P physiological efficiency and higher P requirement.【Conclusion】 The elite high-yielding cultivars, such as Luyuan118, Pumai168 and Huamai7, showed higher yield performance in North-Huanghuai, South-Huanghuai and the middle and lower Yangtze River reaches, respectively. In main wheat production regions of China, increasing shoot biomass and harvest index was the key factor to produce high grain yields. Also, enhancing shoot nutrient uptake and nutrient harvest index was necessary to achieve high grain nutrition quality as well as high yield.

Effect of Continuous Straw Returning on Soil Phosphorus Availability and Crop Phosphorus Utilization Efficiency of Oilseed Rape-Rice Rotation

KunKun WANG,ShiPeng LIAO,Tao REN,XiaoKun LI,RiHuan CONG,JianWei LU

Scientia Agricultura Sinica. 2020, 53(1):  94-104.  doi:10.3864/j.issn.0578-1752.2020.01.009

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【Objective】By exploring the contribution of chemical phosphorus fertilizer and straw returning combined with phosphorus fertilizer to crop productivity, as well as the effect on soil phosphorus availability and phosphorus efficiency under the system of oilseed rape-rice rotation in the Yangtze River, this paper provided a scientific basis for farmland soil phosphorus management.【Method】The experiment was conducted at Huazhong Agricultural University in Wuhan, Hubei Province during the period of 2015-2018. Three treatments in the positioning experiment were selected as followed: (1) phosphorus was not applied (NK); (2) phosphorus application (NPK); (3) phosphorus application and straw returning (NPK+S). By measuring crop yield, phosphorus content and soil Olsen-P, the phosphorus use efficiency of crops was analyzed, and the response of soil Olsen-P to P apparent balance was discussed.【Result】The average yield of oilseed rape and rice under NPK treatment increased by 530.3% and 35.9%, respectively, and the P₂O₅ accumulation increased by 495.3% and 98.5%, respectively, compared with NK treatment. The average yield of oilseed rape and rice under NPK+S treatment increased by 19.1% and 11.0%, respectively, and the P₂O₅ accumulation increased by 20.6% and 11.7%, respectively, compared with NPK treatment. The response of oilseed rape yield and P₂O₅ accumulation to phosphorus fertilizer and straw was better than that of rice. Under the condition of straw returning, the average agronomic efficiency of oilseed rape and rice increased by 6.8% and 33.9%, respectively, and the accumulative phosphorus use efficiency of oilseed rape, rice and anniversary increased by 8.6%, 17.0% and 19.8%, respectively. The effect of straw returning on phosphorus use efficiency and agricultural efficiency of rice was more significant. After four years of oilseed rape-rice rotation, the accumulated deficit of phosphorus in soil was 110.2 kg P₂O₅·hm ^-2 under NK treatment, and the Olsen-P was 1.9 mg·kg ^-1. The accumulated phosphorus surplus of soil under NPK treatment was 210.9 kg P₂O₅·hm ^-2, and the Olsen-P (4.3 mg·kg ^-1) was 126.3% higher than that under NK treatment. NPK+S treatment increased the accumulated phosphorus surplus (222.1 kg P₂O₅·hm ^-2) by 5.3%, and the Olsen-P (5.1 mg·kg ^-1) increased by 18.6% compared with NPK treatment. Straw returning significantly increased the soil Olsen-P, but the soil phosphorus surplus did not increase significantly. Under the condition of continuous straw returning and chemical phosphorus fertilizer application, for every 100 kg·hm ^-2 P surplus of paddy soil, soil Olsen-P under NPK and NPKS treatment increased by 1.8 and 2.0 mg·kg ^-1, respectively. Straw returning promoted soil phosphorus availability.【Conclusion】Phosphorus application significantly increased the yield and P₂O₅ accumulation of oilseed rape and rice, and increased the P surplus and the soil Olsen-P. On the basis of phosphorus application, straw returning further increased the yield and P₂O₅ accumulation of oilseed rape and rice, and improved the use efficiency of phosphorus and agricultural efficiency of crops, especially for rice. At the same time, it could increase the soil Olsen-P while avoiding the excessive accumulation of soil phosphorus.

Responses of Soil Diazotroph Community to Rice Straw, Glucose and Nitrogen Addition During Chinese Milk Vetch Growth

Lu YANG,NaoHua ZENG,JinShun BAI,Xing ZHOU,GuoPeng ZHOU,SongJuan GAO,Jun NIE,WeiDong CAO

Scientia Agricultura Sinica. 2020, 53(1):  105-116.  doi:10.3864/j.issn.0578-1752.2020.01.010

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【Objective】This study was to reveal the regulating roles of carbon (C) sources (rice straw vs. glucose) and nitrogen (N) addition in soil diazotroph community during growth of Chinese milk vetch (Astragalus sinicus L.), which is crucial for the management of crop residue and mineral fertilizer application in Chinese milk vetch - rice rotation system in southern China. 【Method】A pot experiment was conducted with seven treatments, including CK (no exogenous C and N addition), rice straw (Rs) plus various rates of N inputs (Rs, RsN₁ and RsN₂, corresponding to C/N ratios of 66, 25 and 13, respectively), and glucose (Glc) addition plus different N rates (Glc, GlcN₁, and GlcN₂) with same C quantity and C/N ratios in Rs-included treatments. Soils were sampled during the fast-growing phase of Chinese milk vetch, and destined for characterization of nifH gene marker and diazotroph community by using the Illumina Miseq PE300 sequencing and PCR techniques.【Result】Soil C/N ratios were similar between the CK and the treatments with straw or glucose addition alone, while tended to decrease with increasing N input, and significant decrease was observed in the GlcN₂ relative to CK. Regarding to the available nutrients, comparable soil NO₃ ^--N contents were observed under treatments of CK, Rs, and RsN₁, but it was significantly increased by 60% under the RsN₂ treatment. Compared to the CK, Glc-included treatments increased soil NO₃ ^--N content by 35%-79%. There were limited variations of soil available phosphorous (P) content under the CK and Rs-included treatments. 16%-24% decrease of soil available P content was found in the Glc-included treatments than that under CK, but not affected by N rates. The copy number of nifH gene ranged from 80.4×10 ⁶—140.5×10 ⁶ g ^-1soil under all treatments. Compared to the CK, nifH gene copy number tended to increase under treatments with both Rs and Glc addition, while a downward trend was observed with increasing N inputs. Exogenous C and N addition resulted in an overall decrease of diazotroph α-diversity than that under the CK. The responses of diazotroph α-diversity to N supply differed between C sources (Rs vs. Glc). Compared to Rs alone, RsN₁ and RsN₂ had less observed species (decreased by 6%-11%) and Chao 1 index (decreased by 13%-15%), however, GlcN₁ and GlcN₂ enhanced α-diversity to some extent relative to Glc alone. PCoA showed that diazotroph community structure was clustered into different groups depending upon C sources, and was marginally affected by N inputs. Bradyrhizobium was the most abundant genus in all treatments, and its relative abundance was significantly reduced by C and N addition in comparison with CK, however, the magnitude of reduction was obviously less in Rs-included treatments than in Glc-included treatments (12.3%-19.7% vs. 31.6%-40.5%). In contrasting to Bradyrhizobium, the relative abundance of the second most dominant genus (Geobacter) was markedly increased by C addition relative to the CK, with greater magnitude observed in Glc-included vs. Rs-included treatments (by 170%-270% vs. 25.0%-54.6%, respectively). Meanwhile, Multivariate regression tree analysis, RDA, and Mantel analysis revealed that the diazotroph abundance, diversity and community structure were closely associated with soil NO₃ ^--N and available P concentrations. 【Conclusion】The results suggested that effects of N supply on soil diazotroph abundance, diversity and structure were regulated by C sources or the C availability of rice straw and glucose amendments. Meanwhile, the resulted differences of soil available P availability by various C additions might be a key driving factor of reshaping soil diazotroph community during Chinese milk vetch growth.

SPECIAL FOCUS: MOLECULAR BIOLOGY OF CUCUMBER

Molecular Biology of Important Agronomic Traits in Cucumber

ShengPing ZHANG,XingFang GU

Scientia Agricultura Sinica. 2020, 53(1):  117-121.  doi:10.3864/j.issn.0578-1752.2020.01.011

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GWAS Analysis of Hypocotyl Length and Candidate Gene Mining in Cucumber Seedlings

HeXu CAI,KaiLiang BO,Qi ZHOU,Han MIAO,ShaoYun DONG,XingFang GU,ShengPing ZHANG

Scientia Agricultura Sinica. 2020, 53(1):  122-132.  doi:10.3864/j.issn.0578-1752.2020.01.012

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【Objective】The aim of this study was to identify SNP loci and candidate genes significantly correlated with cucumber hypocotyl length trait, which could provide a theoretical basis for revealing the genetic basis and molecular mechanism of cucumber hypocotyl length trait, and lay a foundation for marker-assisted selection breeding of cucumber hypocotyl length trait.【Method】The natural population including 95 cucumber germplasm was employed in this study, and seedlings were grown in the plastic house in Nankou Experimental Field of Chinese Academy of Agricultural Sciences in spring 2016, spring 2017, autumn 2017 and spring 2018, respectively. The hypocotyl length was measured at the two true leaves stage. Structure 2.3.4 software was used to analyze the population structure, and Haploview software was used to analyze the attenuation of linkage imbalance. Then, the whole genome association analysis of hypocotyl length was carried out based on the optimal model. The important candidate genes related to hypocotyl length were predicted according to the LD interval sequence of the associated SNP loci, and the expression pattern of candidate genes were performed by fluorescence quantitative PCR. 【Result】A total of 8 loci, including Hl1.1, Hl1.2, Hl2.1, Hl3.1, Hl3.2, Hl4.1, Hl5.1 and Hl6.1, were detected on Chr. 1, 2, 3, 4 and 5, respectively. Five of them, Hl2.1, Hl3.1, Hl3.2, Hl5.1 and Hl6.1, were detected repeatedly in two or more different environments. By analyzing the LD interval sequences of the associated SNP loci, eight candidate genes, Csa1G074930, Csa1G475980, Csa2G381650, Csa3G141820, Csa4G051570, Csa3G627150, Csa5G174640 and Csa6G362970, were predicted, which were related to cucumber hypocotyl length. Some of the candidate genes involved in regulating plant photomorphogenesis, ubiquitination, and hormone signaling pathway. And some of them were downstream genes regulating cell growth, development and cell size, thus they directly regulated hypocotyl length. Thus, the varied distribution of above genes in different cucumber materials resulted in the different hypocotyl length cucumber germplasm. The organic distribution of polygenes in different cucumber materials formed cucumber germline with different Hypocotyl length. Gene expression analysis showed that Csa1G074930, Csa1G475980, Csa2G381650, Csa4G051570 and Csa5G174640 were highly expressed in short hypocotyl materials and Csa3G141820 and Csa3G627150 were highly expressed in long hypocotyl materials.【Conclusion】Eight SNP loci linked with hypocotyl length, Hl1.1, Hl1.2, Hl2.1, Hl3.1, Hl3.2, Hl4.1, Hl5.1 and Hl6.1, were detected in this study. Eight candidate genes regulating hypocotyl length were predicted, including Csa1G074930, Csa1G475980, Csa2G381650, Csa3G141820, Csa4G051570, Csa3G627150, Csa5G174640 and Csa6G362970.

Genome-Wide Identification of Cucumber ERF Gene Family and Expression Analysis in Female Bud Differentiation

Jian PAN,HaiFan WEN,HuanLe HE,HongLi LIAN,Gang WANG,JunSong PAN,Run CAI

Scientia Agricultura Sinica. 2020, 53(1):  133-147.  doi:10.3864/j.issn.0578-1752.2020.01.013

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【Objective】The objectives of this research were to identify the Ethylene Response Factor (ERF) family genes from cucumber (Cucumis sativus L.) genome and to know the profile of ERF family such as gene number, gene structure and expression characters in cucumber, so as to provide a theoretical basis for exploring what roles the ERF transcription factors played in female flowers development. 【Method】 The ERF genes in cucumber genome were identified by BLAST software in the 9930_V2 genome database based on Arabidopsis ERF genes. EMBOSS, MEME, TBtools, ExPASy and MEGA 7.0 software were used for carrying out various bioinformatics analysis of ERF. The qRT-PCR was used to detect the expression of cucumber ERF gene family in female buds’ differentiation. 【Result】 Total of 138 ERF genes were identified from cucumber genome, which could be divided into 10 classes. These 138 ERF genes were named from CsERF1 to CsERF138, and the number of amino acids of these ERF genes was between 126 and 745. Multiple sequence alignments and Motif analysis showed that the CsERF gene family had two conserved domains, namely AP2/ERF domain and B3 domain. There were 19 ERF genes showed different expression between FFMMAA and ffMMAA genotype, 9 of them up-regulated in FFMMAA genotype and the other 10 up-regulated in ffMMAA genotype. The expression trend analysis showed 31 ERF genes up-regulated and 30 genes down-regulated in the initial-phase of female buds’ differentiation. Furthermore, it was demonstrated that CsERF9 and CsERF31 could bind the GCC-box. 【Conclusion】 138 ERF gene family members were identified from the cucumber 9930_V2 genome. These entire ERF gene shared AP2/ERF domain. Some of the ERF family members were related to sex determination and female flower development and could bind GCC-box to regulate downstream genes expression.

Cloning and Functional Analysis of CsRPL1/2 in Cucumber

WeiYuan SONG,Yu HOU,JianYu ZHAO,XiaoFeng LIU,XiaoLan ZHANG

Scientia Agricultura Sinica. 2020, 53(1):  148-159.  doi:10.3864/j.issn.0578-1752.2020.01.014

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【Objective】AtRPL is an important gene regulating Arabidopsis fruit development and mediating replum formation. Using homologous cloning, the RPL homolog in cucumber was identified, the biological function of CsRPL by expression analysis and ectopic transformation in Arabidopsis was explored.【Method】We cloned the CsRPL gene by performing a BLAST search of AtRPL in cucumber genome. Then we performed amino acid sequence alignment of CsRPL and RPL homologs from other species by MEGA5.2. CsRPL expression pattern in cucumber was detected by real-time quantitative PCR (qRT-PCR) and in situ hybridization. Expression and phenotypic analysis of transgenic Arabidopsis upon ectopic expression of CsRPL were performed as well.【Result】Two RPL genes, named CsRPL1 and CsRPL2, were identified in cucumber with the conserved BELL domain and Homeodomain, and two EAR-Motifs. CsRPL1 was expressed in all organs of cucumber, with the highest expression level in male flowers at anthesis. During early stages of fruit development, the CsRPL1 expression decreased gradually. The expression level of CsRPL2 was significantly lower than that of CsRPL1. In situ hybridization revealed that signals of CsRPL1/2 were detected in the placenta of fruits and the central zone (CZ) of the shoot apical meristem (SAM). Ectopic overexpression of CsRPL1/2 into Arabidopsis resulted in shorter siliques, reduced pollen fertility, and inhibited seed development.【Conclusion】CsRPL1/2 were involved in the development of reproductive organs and might have functional redundancy in cucumber. CsRPL1 might play the primary role under normal growth condition. The function of CsRPL1/2 was not fully conserved as compared to that of AtRPL.

Inheritance and QTL Mapping for Parthenocarpy in Cucumber

ZhiHong NIU,XiaoFei SONG,XiaoLi LI,XiaoYu GUO,ShuQiang HE,LuanJingZhi HE,ZhiHong FENG,ChengZhen SUN,LiYing YAN

Scientia Agricultura Sinica. 2020, 53(1):  160-171.  doi:10.3864/j.issn.0578-1752.2020.01.015

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【Objective】 Parthenocarpy is an important trait affecting both yield and quality in the protected cucumber production. Genetic analysis of parthenocarpy and its QTL mapping are of great significance for improving breeding efficiency in cucumber.【Method】 Based on three-year phenotypic data, the genetic linkage map of cucumber was constructed by using SSR molecular markers obtained from cucumber genome sequencing, and the QTL-Seq analysis was used to map the parthenocarpy of cucumber, using recombinant inbred line F_2:8 constructed from strong parthenocarpic inbred line ‘6457’ and weak parthenocarpic inbred line ‘6426’.【Result】 The inheritance of parthenocarpy in cucumber accorded with quantitative inheritance. A genetic map containing 11 linkage groups was constructed using SSR markers, covering 555.0 cM of the genome with an average distance of 6.8 cM. In the spring of 2016, 2017 and 2018, a single QTL locus related to parthenocarpy was commonly detected between SSR19430 and SSR15419 markers (3.33-5.57 Mb) on chromosome 3, and the genetic distance was 6.6 cM, with 11%, 12.5% and 6.3% contribution rate, respectively. By QTL-Seq analysis, four QTLs related to parthenocarpy were identified on chromosomes 1 (4.38-11.00 Mb), 3 (2.24-10.66 Mb) and 6 (15.67-17.93 Mb; 26.33-27.49 Mb), the QTL on chromosome 3 overlapped with that detected by Map QTL approach. Csa3G047740, Csa3G073810, Csa3G043910 and Csa6G362930 were proposed to be candidate genes associated with parthenocarpic trait in cucumber. 【Conclusion】Four QTLs were identified on chromosome 1, 3 and 6, including one QTL on chromosome 3 that was consistently detected over years with relatively high contribution. The results could facilitate marker-assisted selection and understanding the underlying mechanism of parthenocarpy in cucumber.

Screening and Identification of Cucumber Mutant Resistant to Powdery Mildew

Fei QI,Shu LIN,MengFei SONG,MengRu ZHANG,ShuYan CHEN,NaiXin ZHANG,JinFeng CHEN,QunFeng LOU

Scientia Agricultura Sinica. 2020, 53(1):  172-182.  doi:10.3864/j.issn.0578-1752.2020.01.016

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【Objective】Powdery mildew is one of the most serious diseases that effects cucumber yield and quality. The discovery and research of materials resistant to powdery mildew can fundamentally solve the disease problem. To screen out materials of cucumber resistant to powdery mildew and enrich the breeding population, the Changchunmici mutants were investigated and analyzed in the study.【Method】Totally, 400 Changchunmici mutant lines were inoculated with powdery mildew at seedling stage, and resistant materials were screened preliminarily based on leaf lesion observation combined with disease index analysis. The selected resistant materials were further observed in the natural environment in the field environment. Physiological indexes of resistant materials in the seedling stages were analyzed, including the activities of superoxide dismutase, peroxidase and catalase activity, and physiological indicators such as the contents of chlorophyll a, chlorophyll b, carotenoids and soluble protein. The resistant materials were screened by natural pathogenesis in the field and were further analyzed for the photosynthetic indexes such as transpiration rate, intercellular carbon dioxide concentration, stomatal conductance and net photosynthetic rate. And the contents of ethylene, jasmonic acid and salicylate were determined, and expression of related genes in defense signaling pathways such as ethylene, jasmonic acid, salicylic acid, lignin, and disease-related proteins in leaves were analyzed by real-time PCR. 【Result】Compared with susceptible materials, the resistant materials had less plaque. The net photosynthetic rate and stomatal conductance of resistant materials were higher than those of wild-type Changchunmici, and the intercellular CO₂ concentration of them was lower than that of Changchunmici. In terms of defensive hormones, the contents of ethylene, jasmonic acid and salicylate of resistant materials were higher than those in control. The expression of defense signal related genes in mature leaves of resistant materials was higher compared with susceptible materials. Two powdery mildew resistance materials, Mu-86-2 and Mu-58-9 were screened by inoculation of powdery mildew in seedling stage and natural disease. 【Conclusion】The new materials resistant to powdery mildew could be obtained by screening mutant libraries. The acquisition of these materials had the important value for the genetic research and new varieties breeding of cucumber resistance to powdery mildew.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Effects of Lactobacillus Supplements on Growth Performance of Broilers: A Meta-Analysis

Shuang XING,JingHai FENG

Scientia Agricultura Sinica. 2020, 53(1):  183-190.  doi:10.3864/j.issn.0578-1752.2020.01.017

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Abstract: 【Objective】The purpose of present study was to analyze the effects of Lactobacillus supplements on the growth performance of broilers by meta-analysis. 【Method】A total of 24 articles, including 25 trials and 8 702 subjects, were retrieved from domestic and foreign databases. Reviewing Manager (version 5.3) was used to calculate the standardized mean difference (SMD)(the difference between Lactobacillus group and control group divided by the mean of standard deviation between the two groups). According to the number of samples used in different studies and the standard deviation determined by the indicators, the weights of different research results were determined, and several independent research results were summarized. According to I ² statistics, the heterogeneity of growth performance data was tested, and the publication bias was analyzed by funnel plot and egger test. The random effect model was used to analyze the continuous data for the significant heterogeneity of each index in this analysis and subgroup analysis was carried out according to Lactobacillus strains to explore the effects of different Lactobacillus on the growth performance of broilers.【Result】The analysis showed that Lactobacillus significantly increased the ADG (SMD=1.53, P＜0.001) and reduced the F/G (SMD= -1.50, P＜0.001) of broilers during 0-6 week period of the experiment, and had no significant effect on feed intake (P=0.470). Lactobacillus significantly increased the ADG of broilers (SMD=1.05, P＜0.001) in the growing period, and had a tendency to improve ADG (SMD=0.52, P=0.090) in the finishing period, significantly reduced the growing F/G (SMD= -1.31, P＜0.001) and the finishing F/G (SMD= -0.94, P＜0.001). The growth-promoting effect of Lactobacillus on broilers was better than that the finishing period according to the value of SMD. Funnel plot and egger analysis showed that the bias of F/G (P=0.012) and ADFI (P=0.006) in the early stage reached a significant level, while the biases of other indicators were not significant (P＞0.05). Sensitivity analysis found that random deletion of arbitrary literature data had little effect on the analysis of daily gain and feed-to-weight ratio, indicating that the above results were stable and were not affected by one or several articles. The heterogeneity test found that there was significant heterogeneity in the analysis results of each growth index (P＜0.001), indicating that the results of different literatures differed greatly, possibly due to differences in the Lactobacillus species or feeding doses used in different studies. Subgroup analysis based on Lactobacillus species could reduce the heterogeneity of the meta-analysis. Subgroup analysis showed that L. plantarum promoted the daily weight gain (1.98, P＜0.001) and feed conversion efficiency (-1.66, P＜0.001) on broiler 0-6 week period better than L. casei (0.51, -0.68, P≤0.02) and L. johnsonii (1.15, -0.16, P≤0.02). 【Conclusion】In conclusion, the meta-analysis showed that Lactobacillus could increase the daily weight gain and feed conversion efficiency of broilers at different period, and had no significant effect on feed intake. The promotion effect of Lactobacillus was different, among which L. plantarum was better.

Using Restricted Standardized Linear Regression Model to Estimate Genomic Breed Composition in Composite Breed Animals

Jun HE,Zhi LI,XiaoLin WU

Scientia Agricultura Sinica. 2020, 53(1):  191-200.  doi:10.3864/j.issn.0578-1752.2020.01.018

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【Background】A composite breed is made up of two or more purebreds (ancestries), designed to combine advantageous genetic characteristics from the ancestry breeds and to retain heterosis in future generations without crossbreeding. Unlike crossbred populations, composite variety can be maintained as a purebred. In practice, knowing the ratio of genomic contribution of an ancestry breed to individual composite animals, referred to as the genomic breed composition (GBC), is of importance in animal breed registration, tracing breeding history and population structure, breed conservation, and the prediction of heterosis. Using a set of genomic SNP genotype and an appropriate statistical model, GBC of a purebred or crossbred animal can be estimated. So far, studies on statistical methods devote to the estimation of GBC in composite breed are limited. Linear regression (LR) analysis was commonly used to estimated GBC of individual animals, but it had some limitations such as the coefficients of ancestral breeds does not add to 1.【Objective】The purpose of the present study was to propose and evaluate the use of restricted standardized regression analysis, as an improved approach of linear regression analysis to estimate GBC in composite animals. 【Method】The dataset consisted of 4 323 Beefmaster cattle and purebred animals belonging to their ancestry breeds, namely Brahman, Hereford and Shorthorn. All these animals were genotyped by GeneSeek Genomic Profiling (GGP) bovine 50K SNP chips. Allelic frequencies of each SNP and the Euclidean distance between breeds were computed for the four animal populations, and their genetic relationships were revealed by Hierarchical Clustering based on Euclidean distance of SNP allele frequencies among the four populations. Genomic breed composition of the 4 323 Beefmaster cattle were estimated using RSLR and LR, respectively, based on 7 SNP panels(1K, 5K, 10K, 20K, 30K, 40K, and all the common 47 900 SNP). 【Result】The results of the clustering analysis agreed well with the genetic relationships of Beefmaster and the three ancestral breeds, showing that Beefmaster was more related to Brahman than Herdford and Shorhorn. Linear regression analysis underestimated the genomic contribution ratios of Brahman cattle (0.459-0.462) and shorthorn cattle (0.208-0.212) and at the same time overestimated that of Hereford cattle (0.326-0.333) to Beefmaster cattle. In contrast, estimated GBC of the 4 323 Beefmaster cattle obtained by using RSLR agreed well with expected genomic contribution ratios of the three ancestry breeds, which were 0.497-0.503 for Brahman, 0.262-0.274 for Hereford, and 0.229-0.231 for Shorthorn, respectively. Furthermore, the standard deviations (SD) and coefficients of variance (CV) of GBC obtained by using LR were larger than those obtained using RSLR. With 20K or more SNPs as the reference panels, the SD of GBC estimated by using LR were 0.048 (Brahman), 0.032 (Hereford) and 0.051-0.052 (Shorthorn), and the corresponding CV were 10.46%-10.50% (Brahman), 9.61%-9.76% (Hereford) and 23.94%-25.00% (Shorthorn), respectively. Using RSLR, on the other hand, the SD of GBC pertaining to each of the three ancestry breeds were 0.021 (Brahman), 0.021-0.022(Hereford) and 0.024-0.025 (Shorthorn), and the responding CV were 4.18%-4.20% (Brahman), 7.89%-8.33% (Hereford) and 10.26%-10.68% (Shorthorn), correspondingly. 【Conclusion】The RSLR method provided more accurate and consistent estimates of GBC in the 4 323 Beefmaster cattle than the LR approach. It thus provided a new statistical method for the estimation of GBC in composite animals.

Profiling and Regulation Network of Differentially Expressed Genes During the Development Process of Apis mellifera ligustica Worker’s Midgut

Yu DU,DingDing ZHOU,JieQi WAN,JiaXuan LU,XiaoXue FAN,YuanChan FAN,Heng CHEN,CuiLing XIONG,YanZhen ZHENG,ZhongMin FU,GuoJun XU,DaFu CHEN,Rui GUO

Scientia Agricultura Sinica. 2020, 53(1):  201-212.  doi:10.3864/j.issn.0578-1752.2020.01.019

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【Objective】The whole transcriptome sequencing of Apis mellifera ligustica 7- and 10-day-old workers’ midguts (Am7 and Am10) was previously conducted. In this study, the differential expression profile and regulation network of genes were investigated to reveal the molecular mechanism underlying the midgut development.【Method】Gene expressions were calculated based on FPKM (fragments per kilobase of transcript per million mapped reads) algorithm, and differentially expressed genes (DEGs) were gained following the standard |log₂ fold change|≥1 and P≤0.05. Target mRNAs of ame-miR-6001-3p were predicted utilizing TargetFinder. Annotations of all DEGs in GO and KEGG databases were performed using related software. In addition, DEGs enriched in 13 signaling pathways including AMPK, P13K-Akt, Wnt, cAMP, FoxO, Hippo, mTOR, Jak-STAT, Toll-like receptor, TGF-beta, Notch, MAPK and NF-κB, as well as DEGs targeted by ame-miR-6001-3p were screened out, followed by visualization of enrichment networks and regulation networks with Cytoscape. Finally, Stem loop RT-PCR and RT-qPCR were used to verify the expression and differential expression pattern of ame-miR-6001-3p and DEGs in Am7 and Am10.【Result】A total of 1 038 DEGs were identified in Am7 vs Am10 comparison group, including 515 up- and 523 down-regulated genes, respectively. These DEGs were associated with cellular process, metabolic process and catalytic activity, and significantly enriched in some material and energy metabolisms such as oxidative phosphorylation, amino sugar and nucleotide sugar metabolisms, fatty acid metabolism and purine metabolism, indicative of the active cellular and metabolic activities. Expression cluster analysis suggested that 20, 18, 15 and 14 DEGs were respectively enriched in AMPK signaling pathway, PI3K-Akt signaling pathway, endocytosis and Hippo signaling pathway. In addition, 57 DEGs were enriched in the aforementioned 13 signaling pathways associated with growth and development as well as immune defense, among them one DEG was enriched in several signaling pathways. Moreover, regulation network analysis showed that 54 up-regulated genes and 44 down-regulated genes were targets of ame-miR-6001-3p, respectively; these up-regulated genes were enriched in 43 pathways including inositol phosphate metabolism, Hippo signaling pathway, glutathione metabolism and insulin signaling pathway, while these down-regulated genes were enriched in 20 pathways including Hippo signaling pathway, metabolic pathways, glutathione metabolism and arachidonic acid metabolism. Moreover, RT-qPCR result showed that the variation trend of six randomly selected DEGs were consistent with that in sequencing data, confirming the reliability of DEGs. Finally, ame-miR-6001-3p was definitely expressed and significantly down-regulated in Am10.【Conclusion】In this work, the expression pattern of DEGs and the regulation network between DEGs and ame-miR-6001-3p as well as the potential role of DEGs during the developmental process of A. m. ligustica worker’s midgut were deeply analyzed. The results revealed that the DEGs may participate in various signaling pathways including TGF-beta, Wnt, Hippo, Notch, PI3K-Akt, mTOR, AMPK, NF-κB signaling pathways, thus affecting the growth and development as well as immune defense of the midgut; DEGs were likely to regulate several signaling pathways such as insulin signaling pathway during the midgut development via formation regulation networks with significantly down-regulated ame-miR-6001-3p.

RESEARCH NOTES

Genetic Diversity of Dactylobotrys graminicola and Its Pathogenicity to Hordeum vulgare var. nudum Seedlings

Lin CHEN,RuiMing LIN,FengTao WANG,YunXing PANG,Xue LI,AiPing ZHAO,YanXia ZHANG,JinLing ZHANG,WenXing LI,SuQin HE,Jing FENG,Yun LI,CaiYi WEN,ShiChang XU

Scientia Agricultura Sinica. 2020, 53(1):  213-224.  doi:10.3864/j.issn.0578-1752.2020.01.020

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【Background】In the high-latitude agricultural areas of Qinghai-Tibet Plateau and its peripheral regions, hulless or naked barley (Hordeum vulgare var. nudum) is the only small grain cereal crop in the local area, and it is also the major forage. Dactylobotrys spike blight, caused by Dactylobotrys graminicola, is a new fungal disease only found in the hulless barley growing areas in Qinghai-Tibet Plateau in recent years, which can attack several triticeae crops and cereal grasses and make a great challenge to security of hulless barley production.【Objective】The objective of this study is to research the genetic diversity, systemic evolution of the fungal pathogen of D. graminicola and its effects on the host seed germination, reveal the pathogen epidemics and spread ways, host-pathogen interrelationship and the primary inoculum, and to provide valuable information for disease control.【Method】The diseased plant samples were collected from the disease epidemic areas, and 27 strains were isolated from the typical infected host spike tissues. After identification based on its biological traits of anamorph, the single-conidium strains were cultivated and used for genomic DNA extraction. The whole genomic diversity was evaluated with SRAP markers and strain-specific markers were developed. The evolution relationship between D. graminicola and its related genera or species was analyzed using the two conserved loci of LSU and Rpb2. The conidium suspension was co-cultivated with the germinated or ungerminated hulless barely seeds, and its effect on barley seed germination and seedling growth of hulless barely was analyzed.【Result】Fourteen SRAP primer combinations were selected to detect genetic diversity of D. graminicola strains, and on average, 90 polymorphic DNA bands were produced per primer combination. For D. graminicola strains, only 1.4 strain-specific markers were developed averagely. The strain of Z 13008 derived from common wheat possessed the most 7 SRAP markers. For Fusarium graminearum used as a control strain, 20 specific markers were identified. It was confirmed that there is no close relationship between strain origins of D. graminicola and their genetic diversity. Among the identified strains, only those like Z 13008 from common wheat, Z 13024 from rye and another two (Z 13013 and Z 13006) from hulless barley showed higher differences in genetic diversity compared with the others, indicating that the genetic variation level of D. graminicola population is low. Besides, it is feasible to characterize the 21 D. graminicola strains with a DNA dichotomous fingerprinting key constructed with 20 SRAP markers. D. graminicola is genetically closely related to the fungal species of genus Nectria through phylogenetic analysis. When inoculating seeds with conidial suspensions before germination, the pathogen had no significant suppression on seed germination and growth of seedlings and their roots. While it could infect and restrain seedling root growth when co-cultivating the germinated seeds with conidial suspensions, and resulted in the infected young roots changing into light brown color, but had no significant effect on seeding growth.【Conclusion】D. graminicola showed lower genetic diversity compared with its relative species of F. graminearum, and its genetic variation is closely related to its host plant species. The pathogenicity of the pathogen is weak, and it has no obvious inhibiting effect on the germination and seedling growth of hulless barley.