2023 Vol. 22 No. 2 Previous Issue   
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Crop Science
Analyses and identifications of quantitative trait loci and candidate genes controlling mesocotyl elongation in rice
ZHANG Xi-juan, LAI Yong-cai, MENG Ying, TANG Ao, DONG Wen-jun, LIU You-hong, LIU Kai, WANG Li-zhi, YANG Xian-li, WANG Wen-long, DING Guo-hua, JIANG Hui, REN Yang, JIANG Shu-kun
2023, 22(2): 325-340.  DOI: 10.1016/j.jia.2022.08.080
Abstract ( )   PDF in ScienceDirect  

Rice direct seeding has the significant potential to save labor and water, conserve environmental resources, and reduce greenhouse gas emissions tremendously.  Therefore, rice direct seeding is becoming the major cultivation technology applied to rice production in many countries.  Identifying and utilizing genes controlling mesocotyl elongation is an effective approach to accelerate breeding procedures and meet the requirements for direct-seeded rice (DSR) production.  This study used a permanent mapping population with 144 recombinant inbred lines (RILs) and 2 828 bin-markers to detect quantitative trait loci (QTLs) associated with mesocotyl length in 2019 and 2020.  The mesocotyl lengths of the rice RILs and their parents, Lijiangxintuanheigu (LTH) and Shennong 265 (SN265), were measured in a growth chamber at 30°C in a dark environment.  A total of 16 QTLs for mesocotyl length were identified on chromosomes 1(2), 2(4), 3(2), 4, 5, 6, 7, 9, 11(2), and 12.  Seven of these QTLs, including qML1a, qML1b, qML2d, qML3a, qML3b, qML5, and qML11b, were reproducibly detected in both years via the interval mapping method.  The major QTL, qML3a, was reidentified in two years via the composite interval mapping method.  A total of 10 to 413 annotated genes for each QTL were identified in their smallest genetic intervals of 37.69 kb to 2.78 Mb, respectively.  Thirteen predicted genes within a relatively small genetic interval (88.18 kb) of the major mesocotyl elongation QTL, qML3a, were more thoroughly analyzed.  Finally, the coding DNA sequence variations among SN265, LTH, and Nipponbare indicated that the LOC_Os03g50550 gene was the strongest candidate gene for the qML3a QTL controlling the mesocotyl elongation.  This LOC_Os03g50550 gene encodes a mitogen-activated protein kinase.  Relative gene expression analysis using qRT-RCR further revealed that the expression levels of the LOC_Os03g50550 gene in the mesocotyl of LTH were significantly lower than in the mesocotyl of SN265.  In conclusion, these results further strengthen our knowledge about rice’s genetic mechanisms of mesocotyl elongation.  This investigation’s discoveries will help to accelerate breeding programs for new DSR variety development.

OsMas1, a novel maspardin protein gene, confers tolerance to salt and drought stresses by regulating ABA signaling in rice

WANG Fei-bing, WAN Chen-zhong, NIU Hao-fei, QI Ming-yang, LI Gang, ZHANG Fan, HU Lai-bao, YE Yu-xiu, WANG Zun-xin, PEI Bao-lei, CHEN Xin-hong, YUAN Cai-yuan
2023, 22(2): 341-359.  DOI: 10.1016/j.jia.2022.08.077
Abstract ( )   PDF in ScienceDirect  

Drought and salt stresses, the major environmental abiotic stresses in agriculture worldwide, affect plant growth, crop productivity, and quality.  Therefore, developing crops with higher drought and salt tolerance is highly desirable.  This study reported the isolation, biological function, and molecular characterization of a novel maspardin gene, OsMas1, from rice.  The OsMas1 protein was localized to the cytoplasm.  The expression levels of OsMas1 were up-regulated under mannitol, PEG6000, NaCl, and abscisic acid (ABA) treatments in rice.  The OsMas1 gene was introduced into the rice cultivar Zhonghua 11 (wild type, WT).  OsMas1-overexpression (OsMas1-OE) plants exhibited significantly enhanced salt and drought tolerance; in contrast, OsMas1-interference (OsMas1-RNAi) plants exhibited decreased tolerance to salt and drought stresses, compared with WT.  OsMas1-OE plants exhibited enhanced hypersensitivity, while OsMas1-RNAi plants showed less sensitivity to exogenous ABA treatment at both germination and post-germination stages.  ABA, proline and K+ contents and superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and photosynthesis activities were significantly increased.  In contrast, malonaldehyde (MDA), hydrogen peroxide (H2O2), superoxide anion radical (O2-·), and Na+ contents were significantly decreased in OsMas1-OE plants compared with OsMas1-RNAi and WT plants.  Overexpression of OsMas1 up-regulated the genes involved in ABA signaling, proline biosynthesis, reactive oxygen species (ROS)-scavenging system, photosynthesis, and ion transport under salt and drought stresses.  Our results indicate that the OsMas1 gene improves salt and drought tolerance in rice, which may serve as a candidate gene for enhancing crop resistance to abiotic stresses.

Marker-assisted selection to pyramid Fusarium head blight resistance loci Fhb1 and Fhb2 in a high-quality soft wheat cultivar Yangmai 15
HU Wen-jing, FU Lu-ping, GAO De-rong, LI Dong-sheng, LIAO Sen, LU Cheng-bin
2023, 22(2): 360-370.  DOI: 10.1016/j.jia.2022.08.057
Abstract ( )   PDF in ScienceDirect  

Fusarium head blight (FHB) is one of the most detrimental wheat diseases which greatly decreases the yield and grain quality, especially in the middle and lower reaches of the Yangtze River of China.  Fhb1 and Fhb2 are two major resistance loci against Fusarium graminearum.  Yangmai 15 (YM15) is one of the most popular varieties in the middle and lower reaches of the Yangtze River, and it has good weak gluten characters but poor resistance to FHB.  Here we used Fhb1 and Fhb2 to improve the FHB resistance of YM15 by a molecular marker-assisted selection (MAS) backcrossing strategy.  The selection of agronomic traits was performed for each generation.  We successfully selected seven introgressed lines which carry homozygous Fhb1 and Fhb2 with significantly higher FHB resistance than the recurrent parent YM15.  Three of the introgressed lines had agronomic and quality characters that were similar to YM15.  This study demonstrates that the pyramiding of Fhb1 and Fhb2 could significantly improve the FHB resistance in wheat using the MAS approach

Identification, evolution, expression and protein interaction analysis of genes encoding B-box zinc-finger proteins in maize
XU Xiao-hui, LI Wen-lan, YANG Shu-ke, ZHU Xiang-zhen, SUN Hong-wei, LI Fan, LU Xing-bo, CUI Jin-jie
2023, 22(2): 371-388.  DOI: 10.1016/j.jia.2022.08.091
Abstract ( )   PDF in ScienceDirect  

The B-box (BBX) family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.  BBX proteins play crucial roles in various aspects of plant growth and development, including seedling photomorphogenesis, shade avoidance, flowering time, and biotic and abiotic stress responses.  Previous studies have identified many different BBXs from several plant species, although the BBX family members in maize are largely unknown.  Genome-wide identification and comprehensive analysis of maize BBX (ZmBBX) expression and interaction networks would therefore provide valuable information for understanding their functions.  In this study, 36 maize BBXs in three major clades were identified.  The ZmBBXs within a given clade were found to share similar domains, motifs, and genomic structures.  Gene duplication analyses revealed that the expansion of BBX proteins in maize has mainly occurred by segmental duplication.  The expression levels of ZmBBXs were analyzed in various organs and tissues, and under different abiotic stress conditions.  Protein–protein interaction networks of ZmBBXs were established using bioinformatic tools and verified by bimolecular fluorescence complementation (BiFC) assays.  Our findings can facilitate a greater understanding of the complexity of the ZmBBX family and provide novel clues for unravelling ZmBBX protein functions

Characterization of transgenic wheat lines expressing maize ABP7 involved in kernel development
Zaid CHACHAR, Siffat Ullah KHAN, ZHANG Xue-huan, LENG Peng-fei, ZONG Na, ZHAO Jun
2023, 22(2): 389-399.  DOI: 10.1016/j.jia.2022.08.052
Abstract ( )   PDF in ScienceDirect  

Wheat is one of the major food crops in the world.  Functional validation of the genes in increasing the grain yield of wheat by genetic engineering is essential for feeding the ever-growing global population.  This study investigated the role of ABP7, a bHLH transcription factor from maize involved in kernel development, in regulating grain yield-related traits in transgenic wheat.  Molecular characterization showed that transgenic lines HB123 and HB287 contained multicopy integration of ABP7 in the genome with higher transgene expression.  At the same time, QB205 was a transgenic event of single copy insertion with no significant difference in ABP7 expression compared to wild-type (WT) plants.  Phenotyping under field conditions showed that ABP7 over-expressing transgenic lines HB123 and HB287 exhibited improved grain yield-related traits (e.g., grain number per spike, grain weight per spike, thousand-grain weight, grain length, and grain width) and increased grain yield per plot, compared to WT plants, whereas line QB205 did not.  In addition, total chlorophyll, chlorophyll a, chlorophyll b, and total soluble sugars were largely increased in the flag leaves of both HB123 and HB287 transgenic lines compared to the WT.  These results strongly suggest that ABP7 positively regulates yield-related traits and plot grain yield in transgenic wheat.  Consequently, ABP7 can be utilized in wheat breeding for grain yield improvement

Increasing the appropriate seedling density for higher yield in dry direct-seeded rice sown by a multifunctional seeder after wheat-straw return
TIAN Jin-yu, LI Shao-ping, CHENG Shuang, LIU Qiu-yuan, ZHOU Lei, TAO Yu, XING Zhi-peng, HU Ya-jie, GUO Bao-wei, WEI Hai-yan, ZHANG Hong-cheng
2023, 22(2): 400-416.  DOI: 10.1016/j.jia.2022.08.064
Abstract ( )   PDF in ScienceDirect  
Dry direct-seeded rice (DDR) sown using a multifunctional seeder that performs synchronous rotary tillage and sowing has received increased attention because it is highly efficient, relatively cheap, and environmentally friendly.  However, this method of rice production may produce lower yields in a rice–wheat rotation system because of its poor seedling establishment.  To address this problem, we performed field experiments to determine the rice yield at five seedling density levels (B1, B2, B3, B4, and B5=100, 190, 280, 370, and 460 seedlings m−2, respectively) and clarify the physiological basis of yield formation.  We selected a representative high-quality rice variety and a multifunctional seeder that used in a typical rice–wheat rotation area in 2016 and 2018.  The proportion of main stem panicle increased with increasing seedling density.  There was a parabolic relationship between yield and seedling density, and the maximum yield (9.34−9.47 t ha−1) was obtained under B3.  The maximum yield was associated with a higher total spikelet number m−2 and greater biomass accumulation from heading to maturity.  The higher total spikelet number m−2 under B3 was attributed to an increase in panicle number m−2 compared with B1 and B2.  Although the panicle numbers also increased under B4 and B5, these increases were insufficient to compensate for the reduced spikelet numbers per panicle.  Lower biomass, smaller leaf area, and lower N uptake per plant from the stem elongation stage to the heading stage were partially responsible for the smaller panicle size at higher seedling density levels such as B5.  The higher biomass accumulation under B3 was ascribed to the increases in the photosynthetic rate of the top three leaves m−2 of land, crop growth rate, net assimilation rate, and leaf area index.  Furthermore, the B3 rice population was marked by a higher grain–leaf ratio, as well as a lower export ratio and transport ratio of biomass per stem-sheath.  A quadratic function predicted that 260−290 seedlings m−2 is the optimum seedling density for achieving maximum yield.  Together, these results suggested that appropriately increasing the seedling density, and thereby increasing the proportion of panicles formed by the main stem, is an effective approach for obtaining a higher yield in DDR sown using a multifunctional seeder in a rice–wheat rotation system.

Nitrogen management improves lodging resistance and production in maize (Zea mays L.) at a high plant density

Irshad AHMAD, Maksat BATYRBEK, Khushnuma IKRAM, Shakeel AHMAD, Muhammad KAMRAN, Misbah, Raham Sher KHAN, HOU Fu-jiang, HAN Qing-fang
2023, 22(2): 417-433.  DOI: 10.1016/j.jia.2022.08.074
Abstract ( )   PDF in ScienceDirect  

Lodging in maize leads to yield losses worldwide.  In this study, we determined the effects of traditional and optimized nitrogen management strategies on culm morphological characteristics, culm mechanical strength, lignin content, root growth, lodging percentage and production in maize at a high plant density.  We compared a traditional nitrogen (N) application rate of 300 kg ha–1 (R) and an optimized N application rate of 225 kg ha–1 (O) under four N application modes: 50% of N applied at sowing and 50% at the 10th-leaf stage (N1); 100% of N applied at sowing (N2); 40% of N applied at sowing, 40% at the 10th-leaf stage and 20% at tasseling stage (N3); and 30% of N applied at sowing, 30% at the 10th-leaf stage, 20% at the tasseling stage, and 20% at the silking stage (N4).  The optimized N rate (225 kg ha–1) significantly reduced internode lengths, plant height, ear height, center of gravity height and lodging percentage.  The optimized N rate significantly increased internode diameters, filling degrees, culm mechanical strength, root growth and lignin content.  The application of N in four split doses (N4) significantly improved culm morphological characteristics, culm mechanical strength, lignin content, and root growth, while it reduced internode lengths, plant height, ear height, center of gravity height and lodging percentage.  Internode diameters, filling degrees, culm mechanical strength, lignin content, number and diameter of brace roots, root volume, root dry weight, bleeding safe and grain yield were significantly negatively correlated with plant height, ear height, center of gravity height, internode lengths and lodging percentage.  In conclusion, treatment ON4 significantly reduced the lodging percentage by improving the culm morphological characteristics, culm mechanical strength, lignin content, and root growth, so it improved the production of the maize crop at a high plant density.

Identification of tolerance to high density and lodging in short petiolate germplasm M657 and the effect of density on yield-related phenotypes of soybean
GAO Hua-wei, YANG Meng-yuan, YAN Long, HU Xian-zhong, HONG Hui-long, ZHANG Xiang, SUN Ru-jian, WANG Hao-rang, WANG Xiao-bo, LIU Li-ke, ZHANG Shu-zhen, QIU Li-juan
2023, 22(2): 434-446.  DOI: 10.1016/j.jia.2022.08.047
Abstract ( )   PDF in ScienceDirect  

Soybean yield has been increased through high planting density, but investigating plant height and petiole traits to select for compact architecture, lodging resistance, and high yield varieties is an underexplored avenue to improve yield.  We compared the relationship between yield-related traits, lodging resistance, and petiole-associated phenotypes in the short petiole germplasm M657 with three control accessions over 2017-2018 in four locations of the Huang-Huai region.  The results showed M657 exhibited stable and high tolerance to high planting density and resistance to lodging, especially at the highest density (8×105 plants ha-1).  Regression analysis showed that shorter petiole length was significantly associated with increased lodging resistance.  Yield analysis showed that M657 achieved higher yields under higher densities, especially in the north Huang-Huai region.  There are markedly different responses to intra- and inter-row spacing designs among varieties in both lodging and yield related to location and density.  Lodging was positively correlated with planting density, plant height, petiole length, and number of effective branches, and negatively correlated with stem diameter, seed number per plant, and seed weight per plant.  The yield of soybean was increased by appropriately increasing planting density on the basis of current soybean varieties in the Huang-Huai region.  This study provides a valuable new germplasm resource for introgression of compact architecture traits amenable to high yield in high density planting systems and establishes a high-yield model of soybean in the Huang-Huai region.


SlGH9-15 regulates tomato fruit cracking with hormonal and abiotic stress responsiveness cis-elements

LIN Hao-wei, WU Zhen, ZHOU Rong, CHEN Bin, ZHONG Zhao-jiang, JIANG Fang-ling
2023, 22(2): 447-463.  DOI: 10.1016/j.jia.2022.09.013
Abstract ( )   PDF in ScienceDirect  

Fruit cracking occurs easily during the late period of fruit development when plants encounter an unsuitable environment, dramatically affecting fruit production and marketing.  This study conducted the bulked segregant RNA-Seq (BSR) to identify the key regulatory gene of fruit cracking in tomatoes.  BSR-Seq analysis illustrated that two regions associated with irregularly cracking were located on chromosomes 9 and 11, containing 127 candidate genes.  Further, through differentially expression analysis and qRT-PCR in cracking-susceptible and cracking-resistant genotypes, the candidate gene SlGH9-15 (Solyc09g010210) with significantly differential expression levels was screened.  Bioinformatics analysis of the GH9 gene family revealed that 20 SlGH9 genes were divided into three groups.  The phylogenetic analysis showed that SlGH9-15 was closely related to cell wall construction-associated genes AtGH9B1, AtGH9B6, OsGH9B1, and OsGH9B3.  The cis-acting elements analysis revealed that SlGH9-15 was activated by various hormones (ethylene and ABA) and abiotic stresses.  The expression pattern indicated that 13 SlGH9 genes, especially SlGH9-15, were highly expressed in the cracking-susceptible genotype.  Its expression level gradually increased during fruit development and achieved maximum value at the red ripe stage.  Additionally, the cracking-susceptible tomato showed higher cellulase activity and lower cellulose content than the cracking-resistant tomato, particularly at the red ripe stage.  This study identified SlGH9-15 as a key gene associated with fruit cracking in tomatoes for the first time and gives new insights for understanding the molecular mechanism and complex regulatory network of fruit cracking

An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber
WANG Cui, SUN Jin-jing, YANG Xue-yong, WAN Li, ZHANG Zhong-hua, ZHANG Hui-min
2023, 22(2): 464-470.  DOI: 10.1016/j.jia.2022.08.038
Abstract ( )   PDF in ScienceDirect  

In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.  The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.  Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity.  We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis).  The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.  Moreover, the optimized protocol achieved high signal sensitivity.  The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants

CopE and TLR6 RNAi-mediated tomato resistance to western flower thrips
Jelli VENKATESH, Sung Jin KIM, Muhammad Irfan SIDDIQUE, Ju Hyeon KIM, Si Hyeock LEE, Byoung-Cheorl KANG
2023, 22(2): 471-480.  DOI: 10.1016/j.jia.2022.12.009
Abstract ( )   PDF in ScienceDirect  

The western flower thrips (WFT; Frankliniella occidentalis) is a mesophyll cell feeder that damages many crops.  Management of WFT is complex due to factors such as high fecundity, short reproduction time, ability to feed on a broad range of host plants, and broad pesticide resistance.  These challenges have driven research into developing alternative pest control approaches for WFT.  This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference (RNAi)-mediated silencing of WTF endogenous genes.  For the delivery of RNAi, we developed transgenic tomato lines expressing double-stranded RNA (dsRNA) of coatomer protein subunit epsilon (CopE) and Toll-like receptor 6 (TLR6) from WFT.  These genes are involved in critical biological processes of WFT, and their dsRNA can be lethal to these insects when ingested orally.  Adult WFT that fed on the transgenic dsRNA-expressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples.  In addition, WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts, suggesting that their mortality was likely due to RNAi-mediated silencing of these genes.  Thus, our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F. occidentalis, suggesting that these genes may be deployed to control insecticide-resistant WFT.

Plant Protection
Mitochondrial dynamics caused by QoIs and SDHIs fungicides depended on FgDnm1 in Fusarium graminearum
KANG Jin-bo, ZHANG Jie, LIU Yin-kai, SONG Ji-chang, OU Jian-lin, TAO Xian, ZHOU Ming-guo, DUAN Ya-bing
2023, 22(2): 481-494.  DOI: 10.1016/j.jia.2022.08.118
Abstract ( )   PDF in ScienceDirect  

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating fungal disease on small grain cereal crops, because it reduces yield and quality and causes the mycotoxin contamination to the grain.  Dynamins and dynamin-related proteins (DRPs) are large GTPase superfamily members, which are typically involved in the budding and division of vesicles in eukaryotic cells, but their roles in Fusarium spp. remain unexplored.  Here, we found that FgDnm1, a DRP and homolog to Dnm1 in Saccharomyces cerevisiae, contributes to the normal fungal growth, sexual reproduction and sensitivity to fungicides.  In addition, we found FgDnm1 co-localizes with mitochondria and is involved in toxisome formation and deoxynivalenol (DON) production.  Several quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) cause fragmentated morphology of mitochondria.  Importantly, the deletion of FgDnm1 displays filamentous mitochondria and blocks the mitochondrial fragmentation induced by QoIs and SDHIs.  Taken together, our studies uncover the effect of mitochondrial dynamics in fungal normal growth and how such events link to fungicides sensitivity and toxisome formation.  Thus, we concluded that altered mitochondrial morphology induced by QoIs and SDHIs depends on FgDnm1.

Fatty acid-binding protein gene is indispensable for molting process in Heortia vitessoides (Lepidoptera: Crambidae)
YE Qing-ya, LI Zhi-xing, CHEN Qing-ling, SUN Ming-xu, YIN Ming-liang, LIN Tong
2023, 22(2): 495-504.  DOI: 10.1016/j.jia.2022.08.003
Abstract ( )   PDF in ScienceDirect  

As intracellular fatty acid (FA) carriers, FA-binding proteins (FABPs) widely participate in the absorption, transport, and metabolism of FAs.  It is a key protein in insect lipid metabolism and plays an important role in various physiological activities of insects.  An FABP gene (HvFABP) was cloned from the transcriptional library of Heortia vitessoides Moore (Lepidoptera: Crambidae), and its expression patterns were determined using reverse transcription quantitative PCR (RT-qPCR).  Stage- and tissue-specific expression profiles indicated that HvFABP highly expressed from prepupal to adult stages and in larval midgut and adult wings.  HvFABP expression may be induced through starvation, mRNA expression was downregulated at 24 and 48 h and upregulated at 72 h after starvation.  Furthermore, 20-hydroxyecdysone can induce the upregulation of its expression.  RNA interference-mediated silencing of HvFABP significantly inhibited HvFABP expression, resulting in delayed development, abnormal molting or lethal phenotypes, and a significantly reduced survival rate.  These results indicate that HvFABP plays a key role in the molting of Hvitessoides

Maleness-on-the-Y (MoY) orthologue is a key regulator of male sex determination in Zeugodacus cucurbitae (Diptera: Tephritidae)

FAN Zi-zhen, MA Qin, MA Si-ya, CAO Feng-qin, YAN Ri-hui, LIN Xian-wu
2023, 22(2): 505-513.  DOI: 10.1016/j.jia.2022.08.007
Abstract ( )   PDF in ScienceDirect  

The initiation of sex differentiation in insects is regulated by primary sex determination signals.  In the Medfly Ceratitis capitata and other Tephritids, Maleness-on-the-Y (MoY) is the master gene for male sex determination.  However, the primary signal in Zeugodacus cucurbitae (Coquillett), a very destructive Tephritid pest across the world, remains ambiguous.  In this study, we have isolated and characterized the Medfly MoY homolog in Zcucurbitae, ZcMoY.  ZcMOY protein shows high sequence conservation to its homologs in Bactrocera species.  ZcMoY transcription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults.  Silencing ZcMoY in early embryos by RNAi causes abnormal external genitalia and interior reproductive organs, giving rise to intersexes and feminization of XY individuals.  The expression pattern and knockdown phenotypes of ZcMoY indicate that ZcMoY plays a key role in regulating sex determination of Zcucurbitae males.  Our findings will help the understanding of sex determination in Zcucurbitae and facilitate the development of genetic sexing strains in its biological control.

Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa
WANG Ke, HE Yan-yan, ZHANG You-jun, GUO Zhao-jiang, XIE Wen, WU Qing-jun, WANG Shao-li
2023, 22(2): 514-525.  DOI: 10.1016/j.jia.2022.08.015
Abstract ( )   PDF in ScienceDirect  

Chemosensory proteins (CSPs) perform several functions in insects.  This study performed the gene expression, ligand-binding, and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa, to determine whether EforCSP3 functions in olfaction, especially in host location and host preference.  The results showed that EforCSP3 was highly expressed in the female head, and its relative expression was much higher in adults than in other developmental stages.  The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles, among which dibutyl phthalate, 1-octene, β-elemene, and tridecane had the strongest binding affinity with EforCSP3, besides α-humulene and β-myrcene, and should be assessed as potential attractants.  Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3 possibly involved in volatile binding.  α-Humulene and β-myrcene attracted Eformosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study.  In conclusion, EforCSP3 may be involved in semiochemical reception by Eformosa.

Animal Science · Veterinary Medicine
A 314-bp SINE Insertion in the ZNF2 promoter region may act as a repressor related to regulation of fat deposition in pigs
GU Hao, DU Zhan-yu, Eduard MURANI, Enrico D’ALESSANDRO, CHEN Cai, WANG Xiao-yan, MAO Jiu-de, Klaus WIMMERS, SONG Cheng-yi
2023, 22(2): 526-536.  DOI: 10.1016/j.jia.2022.08.128
Abstract ( )   PDF in ScienceDirect  

Retrotransposons, a type of DNA fragment that can mobilize itself on genome, can generate genetic variations and develop for molecular markers based on the insertion polymorphism.  Zinc finger proteins (ZNFs) are among the most abundant proteins in eukaryotic animals, and their functions are extraordinarily diverse and particularly important in gene regulation.  In the current study, bioinformatic prediction was performed to screen for retrotransposon insertion polymorphisms (RIPs) in six ZNF genes (ZNF2, ZNF3, ZNF7, ZNF8, ZNF10 and ZNF12).  Six RIPs in these ZNFs, including one short interspersed nuclear element (SINE) RIP in intron 1 and one long interspersed nuclear element 1 (L1) RIP in intron 3 of ZNF2, one SINE RIP in 5´ flanking region and one SINE RIP in intron 2 of ZNF3, one SINE RIP in 3´ UTR of ZNF7 and one L1 RIP in intron 2 of ZNF12, were discovered and their presence was confirmed by PCR.  The impact of the SINE RIP in the first intron of ZNF2, which is close to the core promoter of ZNF2, on the gene activity was investigated by dual-luciferase assay in three cell lines.  Our results showed that the SINE insertion in the intron 1 of ZNF2 repressed the core promoter activity extremely significantly (P<0.01) in cervical cancer cells and porcine primary embryonic fibroblasts (HeLa and PEF), thus SINE may act as a repressor.  This SINE RIP also significantly (P<0.05) affected the corrected back fat thickness in Yorkshire pigs.  The corrected back fat thickness of individuals with SINE insertion in the first intron of ZNF2 was significantly (P<0.05) higher than that of individuals without SINE insertion.  In summary, our data suggested that RIPs play important roles in the genetic variations of these ZNF genes and SINE RIP in the intron 1 of ZNF2 may provide a useful molecular marker for the screening of fat deposition in the pig breeding.

The succession of fecal bacterial community and its correlation with the changes of serum immune indicators in lambs from birth to 4 months
YIN Xue-jiao, JI Shou-kun, DUAN Chun-hui, TIAN Pei-zhi, JU Si-si, YAN Hui, ZHANG Ying-jie, LIU Yue-qin
2023, 22(2): 537-550.  DOI: 10.1016/j.jia.2022.08.055
Abstract ( )   PDF in ScienceDirect  

Early bacterial colonization and succession within the gastrointestinal tract have been suggested to be crucial in the development of host immunity.  In this study, we have investigated the changes in live weight and concentrations of selected serum parameters in relation to their fecal bacterial communities as determined by high throughput sequencing of the 16S rRNA gene over the same period in lambs.  The results showed that lambs’ growth performance, the serum parameters, fecal bacterial community and fecal bacterial functions were all affected (P<0.05) by age of the lambs.  Similarity within age groups of fecal microbiota was lower in the preweaning period and increased sharply (P<0.05) after weaning at 60 days.  The similarity between the samples collected from birth to 90 days of age and those collected at 120 days of age, increased (P<0.05) sharply after 30 days of age.  Some age-associated changes in microbial genera were correlated with the changes in concentrations of immune indicators, including negative (P<0.05) correlations between the relative abundance of Lachnospiraceae UCG-010, Eubacterium coprostanoligenes group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-009, Ruminococcaceae UCG-013, Ruminiclostridium 6, Ruminococcaceae UCG-008, and Oscillibacter with serum concentrations of lipopolysaccharide (LPS), D-lactate dehydrogenase (DLA), immunoglobulin (IgA, IgM, and IgG), and cytokines (interleukin-1β (IL-1β), IL-6, IL-12, and IL-17), tumor necrosis factor-α (TNF-α), and the relative abundance of these genera increased from 45 days of age.  In conclusion, these results suggested that the age-related abundances of particular genera were correlated with serum markers of immunity in lambs, and there might be a critical window in the period from birth to 45 days of age which provide an opportunity for potential manipulation of the fecal microbial ecosystems to enhance immune function.

Establishment and evaluation of the primary cultured tibial osteoblast model of broiler chicks

CAO Su-mei, LI Ting-ting, SHAO Yu-xin, ZHAO Yu-zhen, ZHANG Li-yang, LU Lin, ZHANG Ri-jun, HOU Shui-sheng, LIAO Xiu-dong, LUO Xu-gang, WANG Run-lian
2023, 22(2): 551-558.  DOI: 10.1016/j.jia.2022.08.051
Abstract ( )   PDF in ScienceDirect  

Osteoblasts are considered as a major factor contributing to bone development and mineralization, however, few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  Therefore, in the present study, two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  In experiment 1, osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology, alkaline phosphatase (ALP) and alizarin red staining.  Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4, 8, 12, 16, 20, 24, 28 and 32 after incubation, respectively.  The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped, triangular or polygonal morphology.  More than 95% of the cells were stained blue-black after ALP staining, and mineralized nodules were formed after 4 days of continuous incubation.  in experiment 2, lactate dehydrogenase (LDH) activity stayed at a relatively stabilized level although incubation time affected (P=0.0012) it during the whole culture period.  Additionally, incubation time affected (P≤0.0001) the number and proportion of the area of mineralized nodules.  They increased linearly and quadratically (P<0.04) with the increase of incubation time, and remained at a stabilized level from 24 to 32 days of incubation.  The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models (P<0.0001) of the number and proportion of the area of mineralized nodules, respectively.  These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method, and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization, and could maintain a relatively stabilized vitality from 4 to 32 days of incubation; and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.  Therefore, it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus and atypical porcine pestivirus
SONG Xiang-peng, XIA Ying-ju, XU Lu, ZHAO Jun-jie, WANG Zhen, ZHAO Qi-zu, LIU Ye-bing, ZHANG Qian-yi, WANG Qin
2023, 22(2): 559-567.  DOI: 10.1016/j.jia.2022.08.115
Abstract ( )   PDF in ScienceDirect  

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation.  African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.  Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China.  These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry.  They are difficult to be distinguished only by clinical diagnosis.  Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.  In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5´ UTR), African swine fever virus (ASFV) (B646L), and APPV (5´ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.  The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV).  The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL–1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%.  Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively.  The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the  compliance results were the same (100%).  A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively.  This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

The vital role of CovS in the establishment of Streptococcus equi subsp. zooepidemicus virulence
XU Bin, MA Zhe, ZHOU Hong, LIN Hui-xing, FAN Hong-jie
2023, 22(2): 568-584.  DOI: 10.1016/j.jia.2022.08.109
Abstract ( )   PDF in ScienceDirect  

Streptococcus equi subsp. zooepidemicus (SEZ) is an important zoonotic agent.  Here, a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found.  M35246 showed a deletion of 25 contiguous genes as well as a loss-of-function mutation in covS.  Subsequently, a 25-gene-deleted strain (ΔPI), a covS-mutant strain (McovS), and relevant complementary strains were constructed and investigated.  M35246 and McovS were significantly less encapsulated and exhibited poorer anti-phagocytic capacity compared to wild-type SEZ.  McovS was significantly more sensitive to β-lactams, aminoglycosides, macrolides, and lincosamides than wild-type SEZ.  M35246, McovS, and ΔPI exhibited an increase in median lethal dose (LD50) in mice by 105, 105, and 5 times when compared to wild-type SEZ, respectively.  Neither M35246 nor McovS were isolated from mice 48 h after being challenged with approximately 2 000 times the LD50 of wild-type SEZ.  Transcriptome analysis showed that 668 significantly differentially expressed genes existed between McovS and wild-type SEZ.  Numerous virulence factor-encoding genes and anabolic-related genes in McovS that were involved in anti-phagocytosis, capsule formation, pathogenicity, and antibiotic resistance were downregulated significantly relative to the wild-type strain.  This study revealed that the CovS plays a vital role in the establishment of SEZ virulence

Agro-ecosystem & Environment
Different genotypes regulate the microbial community structure in the soybean rhizosphere
QU Zheng, LI Yue-han, XU Wei-hui, CHEN Wen-jing, HU Yun-long, WANG Zhi-gang
2023, 22(2): 585-597.  DOI: 10.1016/j.jia.2022.08.010
Abstract ( )   PDF in ScienceDirect  

The soybean rhizosphere has a specific microbial community, but the differences in microbial community structure between different soybean genotypes have not been explained.  The present study analyzed the structure of the rhizosphere microbial community in three soybean genotypes.  Differences in rhizosphere microbial communities between different soybean genotypes were verified using diversity testing and community composition, and each genotype had a specific rhizosphere microbial community composition.  Co-occurrence network analysis found that different genotype plant hosts had different rhizosphere microbial networks.  The relationship between rhizobia and rhizosphere microorganisms in the network also exhibited significant differences between different genotype plant hosts.  The ecological function prediction found that different genotypes of soybean recruited the specific rhizosphere microbial community.  These results demonstrated that soybean genotype regulated rhizosphere microbial community structure differences.  The study provides a reference and theoretical support for developing soybean microbial inoculum in the future.

Drip fertigation and plant hedgerows significantly reduce nitrogen and phosphorus losses and maintain high fruit yields in intensive orchards
SONG Ke, QIN Qin, YANG Ye-feng, SUN Li-juan, SUN Ya-fei, ZHENG Xian-qing, LÜ Wei-guang, XUE Yong
2023, 22(2): 598-610.  DOI: 10.1016/j.jia.2022.08.008
Abstract ( )   PDF in ScienceDirect  
A field experiment was carried out to evaluate the effects of drip fertigation combined with plant hedgerows on nitrogen and phosphorus runoff losses in intensive pear orchards in the Tai Lake Basin.  Nitrogen and phosphorus runoff over a whole year were measured by using successional runoff water collection devices.  The four experimental treatments were conventional fertilization (CK), drip fertigation (DF), conventional fertilization combined with plant hedgerows (C+H), and drip fertigation combined with plant hedgerows (D+H).  The results from one year of continuous monitoring showed a significant positive correlation between precipitation and surface runoff discharge.  Surface runoff discharge under the treatments without plant hedgerows totaled 15.86% of precipitation, while surface runoff discharge under the treatments with plant hedgerows totaled 12.82% of precipitation.  Plant hedgerows reduced the number of runoff events and the amount of surface runoff.  Precipitation is the main driving force for the loss of nitrogen and phosphorus in surface runoff, and fertilization is an important factor affecting the losses of nitrogen and phosphorus.  In CK, approximately 7.36% of nitrogen and 2.63% of phosphorus from fertilization entered the surface water through runoff.  Drip fertigation reduced the accumulation of nitrogen and phosphorus in the surface soil and lowered the runoff loss concentrations of total nitrogen (TN) and total phosphorus (TP).  Drip fertigation combined with plant hedgerows significantly reduced the overall TN and TP losses by 45.38 and 36.81%, respectively, in comparison to the CK totals.  Drip fertigation increased the vertical migration depth of nitrogen and phosphorus nutrients and reduced the accumulation of nitrogen and phosphorus in the surface soil, which increased the pear yield.  The promotion of drip fertigation combined with plant hedgerows will greatly reduce the losses of nitrogen and phosphorus to runoff and maintain the high fruit yields in the intensive orchards of the Tai Lake Basin.

Agricultural Economics and Management
Optimal design of culling compensation policy under the African swine fever — Based on simulations of typical pig farms in China
GUO Shi-juan, LÜ Xin-ye, HU Xiang-dong
2023, 22(2): 611-622.  DOI: 10.1016/j.jia.2022.12.001
Abstract ( )   PDF in ScienceDirect  
This paper aims to optimize the culling compensation policy from a micro perspective through scenario simulation.  Based on an investigation of 273 pig farms in eight regions, four typical pig farms were constructed according to farm size and breeding mode, representing the swine producers in China.  Besides, a decision objective function of pig farms facing suspected African swine fever (ASF) outbreaks was constructed.  This study used a mathematical programming model to design and simulate scenarios based on compensation standards and local implementation levels, aiming to incentivize pig farms to report epidemics.  The results show that the optimal decisions on epidemic reports differed among typical farms and by herd daily age.  The results suggest the following adjustments for optimizing culling compensation policies: (1) to set culling compensation standards based on the market value and (2) to maintain a high level of epidemic surveillance capability in the animal husbandry and veterinary sector.
The role of time preferences in contract breach: Evidence from Chinese poultry farmers participating in contract farming
HOU Jing, ZHOU Li, Jennifer IFFT, YING Rui-yao
2023, 22(2): 623-641.  DOI: 10.1016/j.jia.2022.12.013
Abstract ( )   PDF in ScienceDirect  

Farmers’ contract breach behavior is cited as one of the major stumbling blocks in the sustainable expansion of contract farming in many developing countries.  This paper examines farmers’ contract breach decisions from the perspective of time preferences.  The empirical analysis is based on a household survey and economic field experiments of poultry households participating in contract farming conducted in Jiangsu Province, China.  A discounted utility model and a maximum likelihood technique are applied to estimate farmers’ time preferences and the effect of time preferences on contract breach in the production and sales phases are explored with a bivariate probit model.  The results show that, on average, the poultry farmers in the sample are generally present biased and impatient regarding future utility.  The regression results show that farmers with a higher preference for the present and a higher discount rate are more likely to breach contracts, and time preferences play a greater role in the production phase than in the sales phase.  When considering heterogeneity, specific investments and transaction costs promote contract stability only for farmers with a low degree of impatience.  Moreover, compared with large-scale farmers, small-scale farmers’ contract breach decisions are more significantly affected by their time preferences.  These results have implications for contract stability policies and other issues that are impacted by the linking of behavioral preferences to agricultural decisions.

Short Communication

Characterization of subunits encoded by SnRK1 and dissection of combinations among these subunits in sorghum (Sorghum bicolor L.)

XIAO Qian-lin, HUANG Tian-hui, ZHOU Chang, CHEN Wei-xi, CHA Jian-kui, WEI Xi-mei, XING Fang-yu, QIAN Meng-ya, MA Qian-nan, DUAN Hong, LIU Zhi-zhai
2023, 22(2): 642-649.  DOI: 10.1016/j.jia.2022.08.068
Abstract ( )   PDF in ScienceDirect  

Sucrose nonfermenting-related protein kinase 1 (SnRK1) is one of the critical serine/threonine protein kinases.  It commonly mediates plant growth and development, cross-talks with metabolism processes and physiological responses to biotic or abiotic stresses.  It plays a key role in distributing carbohydrates and sugar signal transporting.  In the present study, eight SnRK1 coding genes were identified in sorghum (Sorghum bicolor L.) via sequences alignment, with three for α subunits (SnRK1α1 to SnRK1α3), three for β (SnRK1β1 to SnRK1β3), and one for both γ (SnRK1γ) and βγ (SnRK1βγ).  These eight corresponding genes located on five chromosomes (Chr) of Chr1–3, Chr7, and Chr9 and presented collinearities to SnRK1s from maize and rice, exhibiting highly conserved domains within the same subunits from the three kinds of cereals.  Expression results via qRT-PCR showed that different coding genes of SnRK1s in sorghum possessed similar expression patterns except for SnRK1α3 with a low expression level in grains and SnRK1β2 with a relatively high expression level in inflorescences.  Results of subcellular localization in sorghum leaf protoplast showed that SnRK1α1/α2/α3/γ mainly located on organelles, while the rest four of SnRK1β1/β2/β3/βγ located on both membranes and some organelles.  Besides, three combinations were discovered among eight SnRK1 subunits in sorghum through yeast two hybrid, including α1-β2-βγ, α2-β3-γ, and α3-β3-γ.  These results provide informative references for the following functional dissection of SnRK1 subunits in sorghum.