Table of Content

16 October 2021, Volume 54 Issue 20

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

The Genetic Contribution of the Important Breeding Parent Chuanmai 44 to Its Derivatives

LUO JiangTao,ZHENG JianMin,DENG QingYan,LIU PeiXun,PU ZongJun

Scientia Agricultura Sinica. 2021, 54(20):  4255-4264.  doi:10.3864/j.issn.0578-1752.2021.20.001

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【Objective】Common wheat variety Chuanmai 44 has the characteristics of high yield, stable yield and wide adaptability. Ten new varieties have been selected and approved in breeding program using Chuanmai 44 as parent. It indicates Chuanmai 44 is an important breeding parent. To clarify the genetic base of Chuanmai 44 as a vital parent in breeding exercise and identify important genes or QTL within it will be helpful in breeding new elite varieties using Chuanmai 44. 【Method】Fluorescence in situ hybridization was applied to Chuanmai 44 and its ten derived varieties to identify whether there were wheat-alien translocations, and to analyze the chromosome diversity among them. The 660K SNP array data of Chuanmai 44 and its derived varieties were used to calculate the genetic contribution of Chuanmai 44 to its derived varieties and clarify the high transmission genomic segments. Functional molecular markers within cloned genes and linked molecular markers for yield-related traits were used to identify important genes or QTL in Chuanmai 44 for breeding. 【Result】 Chuanmai 44 did not harbor the 6VS/6AL and 1RS/1BL translocation chromosomes which both frequently existed in wheat varieties in Sichuan. Only two out of its ten derivatives, Changmai 32 and Changmai 34, contained 1RS/1BL translocation, which is inherited from another parent Changmai 19. The existence of 1RS/1BL translocation in the two varieties may explain their weak gluten phenotype. Except wheat-relative translocation, the karyotypes of Chuanmai 44 and its 10 derivative varieties also showed polymorphisms on some chromosomes. For instance, there were two types of chromosome 4A among derivatives, and 80% of them showed the same as Chuanmai 44. Chromosomes 5A, 6B and 7B had 4, 2 and 2 karyotypes, respectively. These three chromosomes in the derivative population of Chuanmai 44 showed the same karyotype with Chuanmai 44 in a frequency of 40%. 660K SNP chip analysis identified 1127 genomic segments with high transmission frequency (>50%) within its derived varieties. These genomic segments located on all 21 chromosomes and their mean length was 1.57 Mb. B genome owned the most number and the largest length of the high transmission frequency segments. Chromosomes 4A, 2B and 5B were the three chromosomes with the longest high transmission frequency segments. Chromosomes 4A, 2B and 3B were the three chromosomes with the most number of high transmission frequency segments. Combing the genotype data of 61 functional markers of cloned wheat gene and 13 SNP markers linked with yield-related QTL and the distribution of Chuanmai 44 high transmission genomic regions, we discovered that there are 9 genes markers and 3 QTL markers are anchored in the high transmission rate section of Chuanmai 44. The twelve markers responding to two favorable alleles and three QTL, including TaSdr, NAM-A1, QTKW.sicau-2AS.1, QTKW.Sicau-4AL, QSL.sicau-5AL.2, which exhibited positive effect on preharvest sprouting resistance, effective tiller number, thousand grain weight and spike length, respectively. 【Conclusion】The length of genomic segments retained within its derived varieties was short. It suggested that Chuanmai 44 as a breeding parent had high genetic combining ability, and its chromosomes were easy to recombine with different homologous chromosomes in resulting hybrids, which is beneficial to reduce linkage drag. Therefore, it plays an important role as a skeleton parent in breeding excercise. TaSdr, NAM-A1, QTKW.sicau-2AS.1, QTKW.Sicau-4AL and QSL.sicau-5AL.2 were the important loci in Chuanmai 44, which should be widely used in further breeding program under molecular marker assisted selecting.

IbMKP6, A Mitogen-Activated Protein Kinase, Confers Low Temperature Tolerance in Sweetpotato

JIN Rong,LIU Ming,ZHAO Peng,ZHANG QiangQiang,ZHANG AiJun,TANG ZhongHou

Scientia Agricultura Sinica. 2021, 54(20):  4265-4273.  doi:10.3864/j.issn.0578-1752.2021.20.002

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【Objective】Studying the function of mitogen activated protein kinase (MAPK) IbMPK6 in respect of low temperature stress tolerance in sweetpotato, that will help us to understand the mechanism of adaption to low temperature stress and play a fundamental role in molecular breeding of sweetpotato. 【Method】Agrobacterium tumefaciens strain EHA105 harbored the plasmid 35S::IbMPK6-GFP were transformed intosweetpotato cv. Xushu29 embryogenic callus. Molecular examination and qRT-PCR were used to screen and select transgenic lines. For low temperature stress assay, selected transgenic lines were performed to observe the phenotype and determine the physiological indexes such as Fv/Fm, the content of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) after low temperature treatment and recovery treatment. Diaminobenzidine (DAB) staining and nitro blue tetrazolium (NBT) staining analysis were performed to observe reactive oxygen species (ROS) accumulation. The expression level of the key transcription factor IbCBF3 and downstream gene IbCOR27 involved in low temperature signal transduction pathway were identified before and after low temperature treatment. 【Result】Twelve transgenic lines were generated and three transgenic lines (L3, L8 and L11) with a high expression level of IbMPK6 were selected for low temperature tolerance assay. Under low temperature stress, the level of Fv/Fm in transgenic lines L3, L8 and L11 was 0.79, 0.79 and 0.80, while that in WT was 0.05. After temperature recovery treatment, Fv/Fm in transgenic lines has recovered to former levels, whereas the level of Fv/Fm in WT was only 0.70, which was significantly lower than that in transgenic lines. MDA content of three transgenic (lines L3, L8 and L11) increased by 0.02, 0.04 and 0.02 μmol·g^-1, and it of WT increased by 0.05 μmol·g^-1 after low temperature stress treatment, respectively. After recovery treatment, MDA content in transgenic lines was 0.01 μmol·g^-1 on average, whereas it of WT was 0.03 μmol·g^-1. The results of DAB and NBT staining showed that the leaves of WT were stained deeper than those of transgenic lines, indicated that hydrogen peroxide and superoxide anion were accumulation less in transgenic lines than in WT. Furthermore, H₂O₂ level in WT was significantly higher than that in transgenic lines under low temperature stress condition and after recovery treatment. Low temperature regulated the expression level of IbCBF3 and IbCOR27 genes, but the expression level in transgenic lines was higher than that in WT. 【Conclusion】Overexpression of IbMPK6 in sweetpotato resulted in enhanced tolerance to low temperature stress, via alleviating the damage of membrane and photosynthetic system, and decreasing ROS accumulation. IbMPK6 involved in low temperature signaling transduction pathway by up-regulating the expression level of cold related genes IbCBF3 and IbCOR27.

Effects of PvEG261 Gene on the Fusarium Wilt and Drought- Resistance in Common Bean

XUE RenFeng,FENG Ming,HUANG YuNing,Matthew BLAIR,Walter MESSIER,GE WeiDe

Scientia Agricultura Sinica. 2021, 54(20):  4274-4285.  doi:10.3864/j.issn.0578-1752.2021.20.003

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【Objective】By analyzing the sequence and expression pattern characteristics of PvEG261 from common beans, and studying its resistance to Fusarium wilt and drought, the foundation was laid for the signal regulation network analysis of Fusarium wilt and drought-resistance and molecular breeding in common beans. 【Method】 Bioinformatics analysis was performed on the open reading frame (ORF) of PvEG261 to predict the physical and chemical properties, secondary structure, signal peptide sequence of the protein encoded by the PvEG261, and search for highly homologous protein sequence in NCBI database through Blastp tool online for sequence alignment and phylogenetic tree construction; the tissue expression specificity of PvEG261 and the expression pattern in response to Fusarium wilt pathogen and drought stress were analyzed by qRT-PCR; PvEG261 overexpression vector was constructed and transformed into Agrobacterium rhizogenes K599 to induce the generation of hairy transgenic roots in common beans. Meanwhile, the PvEG261 silencing vector was constructed, and the transcription product in vitro was inoculated on the seedlings of common bean to interfere with PvEG261 expression. Through inoculation with the pathogen and drought treatment, the phenotypes of control, PvEG261-overexpressed and silenced plants were observed, disease and drought-resistance were both identified, and hydrogen peroxide (H₂O₂) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and peroxidase (POD) activity as physiological and biochemical indicators were all assayed. 【Result】 The cDNA sequence of PvEG261 was 471 bp, which encodes a protein composed of 156 amino acids. The structure prediction indicated that it contained 10 strand structures, the predicted molecular mass of the encoding product was 38.89 kD, and the theoretical pI was 5.21. PvEG261 belonged to the members of dirigent gene superfamily, it contained a signal peptide sequence of 10 amino acids, and belonged to a secreted protein. The relationship between PvEG261 and cowpea DIR22 protein is the closest, which reached 91.61%. The results of qRT-PCR showed that the expression in the root tissues increased significantly after inoculation with Fusarium wilt pathogen and drought treatment, and the gene has obvious tissue expression specificity, with the highest expression level in the roots. After inoculation with pathogen and drought treatment, the disease and drought-resistance of the overexpressed plants were significantly improved in comparison with the control, the plant disease scores and the wilting degree caused by water shortage were significantly reduced, and the H₂O₂ content, POD and SOD activity in the roots were all significantly higher than the control plant, while the MDA content was dramatically lower than the control plant. The disease and wilting degree of the gene silenced plants were significantly increased. The H₂O₂ content, POD and SOD activity in the roots were significantly lower than the control plant, and the MDA content was significantly higher than the control plants. 【Conclusion】 PvEG261 responded to Fusarium wilt pathogen infection and drought stress, and positively regulated the Fusarium wilt and drought-resistance in common beans.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

New Development of Modern Cotton Farming Theory and Technology in China - Concentrated Maturation Cultivation of Cotton

NIE JunJun,DAI JianLong,DU MingWei,ZHANG YanJun,TIAN XiaoLi,LI ZhaoHu,DONG HeZhong

Scientia Agricultura Sinica. 2021, 54(20):  4286-4298.  doi:10.3864/j.issn.0578-1752.2021.20.004

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Concentrated maturation is the basic requirement of cotton mechanical harvesting, referring to the phenomenon that bolls of the whole cotton plant complete opening in a relatively short period of time. Concentrated maturation cultivation refers to the cultivation management technique and measure for achieving optimized fruiting and grouped maturation in cotton. After many years of research and practice, the theory and technology of centralized maturation cultivation of cotton in China have been established, becoming an important approach of modern cotton farming theory and technology. The concept and connotation, key regulatory technologies and the eco-physiological mechanisms of cotton concentrated maturation were innovatively highlighted and reviewed in this paper. The cultivation and regulation for cotton concentrated maturation should start from sowing. The precision monoseeding technology assured well-established strong seedlings, which created a solid basic population for realization of concentrated maturation. Based on good stand establishment, irrigation, fertilization and plant growth regulators were comprehensively used to regulate the growth and development of plant individuals and populations to construct the ideal plant type and the efficient population structure according to local ecological conditions and production conditions, and finally realize the optimized spatiotemporal distribution and concentrated opening of cotton bolls. Precision monoseeding created a suitable soil pressure to seedlings during emergence and a dark environment before emergence, and induced expression of apical hook formation- and hypocotyl elongation-related genes, which promoted hook formation and hypocotyl growth and seedling emergence. The seedlings under monoseeding had independent growth space after emergence, which had little mutual influence and were easy to establish strong seedlings. Close-planting and chemical control inhibited the photosynthesis of leaves sourced from vegetative branches, and altered the content and distribution of endogenous hormones through changing the expression of key genes of hormone metabolism, which regulated the vegetative branching and apical growth of main stems, and finally realized non-pruning and promoted concentrated boll-setting. Leaf-derived jasmonic acid induced by partial root-zone irrigation, as a long-distance signal transported through the phloem to the irrigated root side, promoted the expression of aquaporin gene, and then improved the capacity of water absorption and water use efficiency. The partitioning of assimilates to cotton bolls and the defoliation rate were significantly improved by fertigation under partial root-zone drip irrigation. In this case, reduced inputs of water and fertilizer as well as significant reduction in heterozygosity in machine harvested seed cotton have been achieved without yield loss. The theory and technology of cotton concentrated maturation cultivation was a new achievement of cotton cultivation research in the new era, and an important scientific and technological support for the development of modern cotton industry. In order to provide a more powerful theoretical and technical support for light and efficient cultivation of cotton in the future, on the one hand, in-depth study is required to reveal the physiological and ecological mechanisms of concentrated maturation. On the other hand, it is necessary to innovate the key cultivation techniques, and to develop the well-matched varieties and agricultural mechanic equipment for their better integration. It is also necessary to strengthen the combination of agronomic technologies with smart agricultural technology, so as to provide a more powerful theoretical and technical support for concentrated maturation cultivation of cotton.

Remote Sensing Estimation of Cotton Biomass Based on Parametric and Nonparametric Methods by Using Hyperspectral Reflectance

ZHOU Meng,HAN XiaoXu,ZHENG HengBiao,CHENG Tao,TIAN YongChao,ZHU Yan,CAO WeiXing,YAO Xia

Scientia Agricultura Sinica. 2021, 54(20):  4299-4311.  doi:10.3864/j.issn.0578-1752.2021.20.005

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【Objective】The aim of this experiment was to use hyperspectral remote sensing data to estimate cotton biomass quickly and non-destructively, and to assess the performance differences between parameterized and non-parametric algorithms on cotton. 【Method】This experiment was based on the dataset of four cotton varieties in two years (2004 and 2005), and the two-year data were modeled and verified respectively. The biomass estimation models in different periods (before and after boll opening) were built by utilizing parameterized algorithms, including vegetation index method and continuous wavelet transform, and non-parameterized algorithms, including partial least squares regression, random forest, artificial neural network, regression tree, bag tree and enhanced tree, support vector machine and Gaussian process regression, respectively. 【Result】Near-infrared and red edge bands were still the most effective bands in monitoring cotton biomass of remote sensing. The parameterized method was simple, efficient and accurate. Among the parametric methods, CI_{red edge} was proved to be the best vegetation index for cotton biomass estimation with high independent verification results (before boll opening: RMSE=27.23 g·m^-2; after boll opening: RMSE=48.81 g·m ^-2). The result based on continuous wavelet transform alleviated the underestimation phenomenon of vegetation index, especially after boll opening (before boll opening: RMSE=31.54 g·m ^-2; after boll opening: RMSE=37.57 g·m ^-2). Among the non-parametric methods, the random forest was the best algorithm for cotton biomass estimation (before boll opening: RMSE=20.48 g·m ^-2; after boll opening: RMSE=30.28 g·m ^-2). The estimation accuracy of the two types of algorithms was affected by cotton wool, and the estimation accuracy after boll opening was significantly lower than before boll opening. 【Conclusion】In this study, the cotton biomass estimation models based on parameterized and non-parameterized algorithms were evaluated, and it was proved that the non-parameterized algorithm had high inversion accuracy and could be used as an important method for non-destructive monitoring of cotton biomass.

Effects of Black Film Mulched Ridge-Furrow Tillage on Soil Water- Fertilizer Environment and Potato Yield and Benefit Under Different Rainfall Year in Semiarid Region

YANG FengKe,HE BaoLin,DONG Bo,WANG LiMing

Scientia Agricultura Sinica. 2021, 54(20):  4312-4325.  doi:10.3864/j.issn.0578-1752.2021.20.006

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【Objective】To explore the effects of annual precipitation regime and ridge shapes interaction on soil water and fertilizer environment and potato yield and efficiency of black film mulched ridge-furrow tillage, to analyze and explain the mechanism of water and fertilizer interaction to promote crop production and efficient water use, so as to provide theoretical basis for deeply exploring the yield increase potential of the film mulched ridge-furrow tillage technique. 【Method】Based on the technology of black plastic film mulched ridge-furrow tillage with micro-rainwater catchment ditches on the ridges, we developed three types of soil water-fertilizer regulating tillage systems that consists of low ridge (high 16 cm)-furrow (wide 40 cm), middle ridge (high 24 cm)- furrow (wide 40 cm) and high ridge (high 32 cm)-furrow (wide 40 cm) tillage, all with a 10 cm high and 20 cm wide rainwater catchment ditch on ridges, named as BLRF, BMRF and BHRF, respectively, and used as main treatments. Then, using the local custom white film mulched ridge（high 16 cm, wide 60 cm）-furrow (wide 40 cm) tillage (WRF) as control, a 3-year consecutive field experiments of four treatments had been conducted from 2016 to 2018. Soil water content of 0-200 cm soil layer in the potato key growth period of sowing, seedling, budding, flowering, tuberization and maturing as well as the content of soil organic carbon (SOC), nitrogen, phosphorus and potassium and their available component (TN, TP, TK and AN, AP, AK) of 0-30 cm soil layer at the end of the experiment period were determined, soil water storage and water utilization efficiency were calculated, the correlation between soil water, fertilizer and potato yield were analyzed. 【Result】Regardless of the precipitation years, both BLRF, BMRF, BHRF and WRF tillage had caused crop increase consumption of soil water in 40-120 cm soil layer. The soil water content and water storage (SWS) in 0-200 cm soil layers in the potato six key growth stages for BHRF, BMRF and BLRF were significantly (P<0.05) higher than those for WRF. Sufficient precipitation and the increased soil water harvested via black film mulched ridge-furrow tillage with micro-rainwater catchment ditches on ridges significantly complement the soil water in 120-200cm soil layer and effectively maintained soil water balance interseason. Take rainfall patterns into account, the effects of soil water conservation for BLRF and BMRF are better than that for BHRF in dry and normal year, and that for the three tillage models were significant efficient in wet year, all were significantly better than that of WRF. Meanwhile, the content of examined soil nutrient parameters in 0-30 cm soil layer under BHRF, BMRF and BLRF at the end of the experiment period were uniformly significantly higher than that of WRF (P<0.05),with the average content of TN,TK,AN, AP and AK were increased by 4.5%-5.6%, 3.6%-5.9%, 8.4%-18.4%, 15.3%-22.3% and 7.1%-13.3% except that of SOC and TP, respectively. In addition, the potato yield, water use efficiency (WUE), net income and output/input ratio for BLRF, BMRF and BHRF were all significantly higher than those for WRF, mainly contributed to the increased the number and weight of large potatoes caused by the improved soil water and nutrient condition. On 3 year average, the yield, WUE, net income and output/input ration of potato were increased by 16.9%-19.0%、15.5%-19.2%、23.3%-27.3% and 12.1%-18.2%, respectively. The four benefit parameters were more sounded under BLRF and BMRF in dry and normal year and highly significant under all the three tillage patterns in wet year. We observed that the 3-year average potato yield positively significant correlation to the contents of TN, TP, TK AN, AP, AK and highly negative correlation to the 3-year average water consumption (ET). Path analysis indicated that AP, ET, potato growth period precipitation (GPR), TK and TN had explained 99.4% of the yield variation. 【Conclusion】The superimposed water collection effect of black film mulched ridge furrow tillage with micro-rainwater catchment ditches on the ridges significantly improves the soil moisture condition, which, in turn, great promoting the vigorous growth of potato and led more organic matter (stems, roots, etc.) return to the soil,then the nutrients from the decomposition of the organic matter and the applied fertilizer improves the soil nutrient content. Good soil water and fertilizer conditions effectively improve the mutual relationship between soil water and fertilizer, increased the supply of water and fertilizer, and significantly improved the potato yield, WUE, net income and input/output ratio. BLRF and BMRF performed well in dry and normal years, BLRF, BMRF and BHRF performed well in wet years, while BLRF and BMRF performed well in all precipitation years. Therefore, BLRF and BMRF are the two most efficient cultivation model after WRF for dry potato high yield production in semi-arid regions of China.

PLANT PROTECTION

Molecular Variation and Phylogenetic Relationship of Apple Scar Skin Viroid in Seven Cultivars of Apple

LI ZiTeng,CAO YuHan,LI Nan,MENG XiangLong,HU TongLe,WANG ShuTong,WANG YaNan,CAO KeQiang

Scientia Agricultura Sinica. 2021, 54(20):  4326-4336.  doi:10.3864/j.issn.0578-1752.2021.20.007

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【Objective】The objective of this study is to explore the molecular variation and phylogenetic relationship of apple scar skin viroid (ASSVd) in different cultivars of apple, and to lay a foundation for further revealing the molecular variation mechanism of ASSVd. 【Method】The different cultivars of apple including Fuji, Tonami, Wang Lin, Mid-Autumn King, Gold Delicious, Xin Nonghong and Xin Nonghuang, infected by ASSVd were used as materials to amplify complete genome sequence by specific primers, and then molecular cloning and sequencing were performed. Biological software DNAMAN was used to analyze the identities of the variant sequences and the MEGA was used to construct a phylogenetic tree. 【Result】A total of 210 sequences of ASSVd were obtained, with a total of 17 variants, with a size of 325-333 nt. Seventeen variants of ASSVd were aligned and analyzed with other published representative isolates. The result showed that all the variants were divided into three groups. Group I includes 10 variants, which are closely related to the reported Baoding Fuji isolate (KR264032.1). Group II includes 6 variants, which are clustered separately. Group III includes one variant, which is closely related to the Xinjiang Fuji isolate (EU031455.1). Based on 8 variation base sites at genome 0-3, 221, 251, 284, and 302, the 17 variants were divided into 6 type including: 1. nt 0-3 (GGTA) + nt 41-46 (TAAAAT) + nt 221 (T) + nt 251 (T) +nt 284 (G) + nt 302 (T); 2. nt 0-3 (XGGT) + nt 41-46 (AGATAX) + nt 221 (T) + nt 251(X) + nt 284 (A) + nt 302 (A); 3. nt 0-3 (XGGT) + nt 41-46 (AGATAX) + nt 221 (X) + nt 251 (T) + nt 284 (A) + nt 302 (A); 4. nt 0-3 (XGGT/GGTA) + nt 41-46 (AGATAX) + nt 221 (T) + nt 251 (T) + nt 284 (A) + nt 302 (A); 5. nt 0-3 (GGTA) + nt 41-46 (TAAAAT) + nt 221 (X) + nt 251 (G) + nt 284 (G) + nt 302 (T); 6. nt 0-3 (GGTA) + nt 41-46 (TAAAAT) + nt 221 (T) + nt 251 (G) + nt 284 (G) + nt 302 (T). X represents missing. There are 6 types of variants in Fuji, type 4 (47.5%) is the main type. There are 3 types of variants in Wang Lin, type 5 (43%) is the main type. There are 3 types of variants in Gold Delicious, type 4 (60%) is the main type. ASSVd obtained from Tonami is type 5 (100%). ASSVd obtained from Mid-Autumn King is type 4 (100%). There are 2 types in Xin Nonghong, type 1 (83.3%) is the main type. There are 3 types in Xin Nonghuang, type 1 (62.5%) is the main type. 【Conclusion】Seventeen variants from 7 cultivars are divided into 6 types according to the 8 variation base sites of ASSVd nt 0-3, 221, 251, 284, and 302. Different apple cultivars carry different ASSVd population structures. The types and proportions of ASSVd variants are different.

The Detection of Citrullus lanatus Cryptic Virus Using TaqMan-qPCR Method

ZHAO LiQun,QIU YanHong,ZHANG XiaoFei,LIU Hui,YANG JingJing,ZHANG Jian,ZHANG HaiJun,XU XiuLan,WEN ChangLong

Scientia Agricultura Sinica. 2021, 54(20):  4337-4347.  doi:10.3864/j.issn.0578-1752.2021.20.008

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【Objective】Citrullus lanatus cryptic virus (CiLCV) is an important seed-transmitted virus that newly-emerging in watermelon in recent years. The objective of this study is to develop a detection method for CiLCV with real-time fluorescent quantitative PCR with TaqMan probes (TaqMan-qPCR), and to provide technical supports for the CiLCV detection from seeds and seedlings, and also for disease controlling in the future. 【Method】The CiLCV was found in Beijing watermelon producing area with high-throughput sequencing based on small RNAs. The full sequence of CiLCV-dsRNA1 and CiLCV-dsRNA2 was cloned and specific amplifying primers were designed to set up the detection method of the RT-PCR and TaqMan-qPCR. The other eight common viruses (cucumber green mottled mosaic virus, cucumber mosaic virus, Cucumis melo endornavirus, cucurbit chlorotic yellows virus, squash mosaic virus, tomato spotted wilt virus, watermelon mosaic virus, zucchini yellow mosaic virus) were used as control to analyze the method specificity. The standard curve of CiLCV was performed to evaluate the method sensitivity. Furthermore, the novel detection methods of TaqMan-qPCR and RT-PCR were used to inspect CiLCV in watermelon, cucumber, melon, and rootstock pumpkin seedlings that randomly collected from the main producing areas of cucurbits crop in North China. 【Result】The small RNAs data with high-quality were obtained, and 17 assembled contigs were found to share homology with CiLCV genomes after data analysis. The full-length of CiLCV-dsRNA1 and CiLCV-dsRNA2 was cloned with 1 603 and 1 466 nt in length, respectively, sharing the highest nt sequence identity (about 99.4% and 99.8%, respectively) with CiLCV that isolated from Henan Province (GenBank number KY081285, KY081284). The established RT-PCR for CiLCV showed a single amplified band, while the novel TaqMan-qPCR for detecting CiLCV showed good sensitivity and specificity, and could detect about 2×10³ copies of CiLCV. The detection sensitivity of TaqMan-qPCR was about 100 times higher than that of RT-PCR. In addition, only one Beijing-watermelon sample was CiLCV positive, while the rest watermelon, melon, cucumber, and rootstock pumpkin seedlings were CiLCV negative, indicating that the virulence of seedlings in the main producing areas of cucurbits crop in China was not high. 【Conclusion】The established TaqMan-qPCR method for CiLCV detection has high specificity and sensitivity, and is suitable for rapid detection and accurate identification of CiLCV at ports and laboratories. In view of CiLCV can spread rapidly through seeds and seedlings, China should pay more attention to the detection of virulent seeds and seedlings to prevent the virulent seeds and seedlings flowing into the production process and then being transported and spread to the whole country, which may cause great economic losses to the industry.

Cloning, Expression and Anti-Virus Function Analysis of Solanum lycopersicum SlN-like

LIU ChangYun,LI XinYu,TIAN ShaoRui,WANG Jing,PEI YueHong,MA XiaoZhou,FAN GuangJin,WANG DaiBin,SUN XianChao

Scientia Agricultura Sinica. 2021, 54(20):  4348-4357.  doi:10.3864/j.issn.0578-1752.2021.20.009

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【Objective】As an important vegetable crop, tomato (Solanum lycopersicum) is endangered by various biological factors including pests, fungi, bacteria and viruses. The objective of this study is to clarify the antiviral function and mechanism of S. lycopersicum resistance gene SlN-like, and to provide a theoretical basis for the genetic breeding of antiviral S. lycopersicum and the targeted development of the antiviral agents. 【Method】The full length of SlN-like was obtained from the Solanaceae Genomics Network database and was divided into four segments, fusion PCR was used to amplify the full length of sequence. Bioinformatics was used to analyze the evolutionary relationship, protein characteristics, conserved domains, subcellular location and interaction relationship of SlN-like. Real-time fluorescent quantitative PCR was used to analyze the SlN-like expression in S. lycopersicum roots, stems, leaves, flowers and fruits and its response after tobacco mosaic virus (TMV) infection. S. lycopersicum endogenous SlN-like was silenced using tobacco rattle virus (TRV)-mediated gene silencing technology, and the silent plants were inoculated with TMV-GFP to clarify the influence of SlN-like on virus infection. The expressions of abscisic acid (ABA), jasmonic acid (JA) and ethylene (ET) hormone-related genes in silenced plants, and the expression of SlN-like after application of ethephon (ETH) for 3, 6, 12 and 24 h were analyzed by real-time fluorescence quantitative PCR to investigate the mechanism of SlN-like regulatory hormone pathway in response to virus infection. 【Result】Through molecular cloning and fusion PCR technology, a 3 444 bp SlN-like was cloned from S. lycopersicum variety Micro-Tom, and uploaded to NCBI to obtain the sequence number MW792493. Through bioinformatics analysis, it was found that SlN-like contains TIR, NB-ARC and NACHT domains, and is closely related to Solanum tuberosum N-like (AAP44394.1). SlN-like expressed in all tissues of S. lycopersicum, with the highest expression in stems, followed by roots, flowers, leaves and fruits. After TMV-GFP infection S. lycopersicum at 5th and 7th day, the SlN-like expression level was higher than that of PBS treatment, and TMV-GFP infection would cause the expression of SlN-like to increase continuously. TRV vector induced silencing of SlN-like in S. lycopersicum, and it was found that silencing 78.3% of SlN-like did not affect tomato growth phenotype, but silencing SlN-like promoted the infection of TMV-GFP. Real-time fluorescent quantitative PCR analysis found that the expression of ERF1 in SlN-like-silent plants was significantly reduced, only 12.5% of that in the control group. The expression of SlN-like increased after 3 h of external application of ethephon, and reached the highest peak at 12 h, which was 2.71 times that of the control group, and returned to normal at 24 h. 【Conclusion】S. lycopersicum SlN-like belongs to the NBS-LRR disease-resistant protein family, its expression is induced by TMV infection. Silencing SlN-like can promote TMV-GFP infection and reduce the expression of ethylene-related gene ERF1, while external application of ethephon resulted in the differential expression of SlN-like, revealing that SlN-like participates in S. lycopersicum antiviral defense through the ethylene pathway.

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

Theoretical Connotations and Pricing Mechanisms for Agricultural Ecological Compensation Within the Context of Green Development

ZHOU Ying,MEI XuRong,YANG Peng,LIU Jing

Scientia Agricultura Sinica. 2021, 54(20):  4358-4369.  doi:10.3864/j.issn.0578-1752.2021.20.010

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【Objective】Within the context of green development, the research on mechanisms for agricultural ecological compensation has become an extremely urgent and strategic task for the protection of the ecological environment in functional agricultural areas in China. The standards for ecological compensation directly affect the actual effect of policy internalization and externality. The redefine the theoretical connotations of agricultural ecological compensation, determine the concepts and basis of compensation pricing, and are the effective way to improve the accuracy and directivity of subsidy policy. 【Method】Using normative analysis as well as inductive and deductive methods, the boundary of green development oriented agricultural ecological compensation policy was re-positioned, and the types and content of agricultural ecological compensation were systematically divided, in an attempt to develop a pricing mechanism that focused on the measurement of external effects and scientifically answer the core questions of "why compensate?" and "what to compensate?" in the field of agricultural ecological compensation. 【Result】Firstly, the connotations of ecological compensation for agricultural green development were scientifically analyzed: ecological compensation for agricultural green development is an environmentally friendly and economic means of producing spillover benefits (costs) internalization when protecting agricultural resources and the environment. The core content of compensation includes two parts: the protection compensation for agricultural resource assets, and the compensation for green agricultural production behavior. Secondly, According to the environmental impact of technology on the ecosystem, the compensation content can be divided into four types: (1) the compensation for resource development and construction, aiming at the reduction of positive externality in the environment and the loss of private interests; (2) the compensation for resource protection and utilization, aiming at a reasonable return for the preservation and proliferation of ecological capital; (3) the compensation for environmental pollution and protection, aiming at reducing the costs and losses from negative externality in the environment; (4) the compensation for environmental quality improvement, aiming to compensate for investment and income losses from improving the positive externality of the environment. Thirdly, the accounting basis of compensation standards for these four types of compensation was developed, among these standards: the compensation for resource development and construction should be based on the replacement cost of resource production or maintenance, the expected return of resource assets, and the opportunity cost of development; the compensation for resource protection and utilization should be based on the direct investment of resource protectors, the opportunity cost of development, and the ecological service value of resource protection; environmental pollution control and quality improvement belong to compensation for the application of green technology, and its compensation pricing is mainly based on the additional production costs, environmental costs, development opportunity cost, and technology spillover benefit value. 【Conclusion】The innovation of an agricultural ecological compensation system guided by green development in China includes: first, which should, on the one hand, divide the policy boundary of agricultural ecological compensation from the perspective of the environmental contribution of green production, and on the other hand, should compensate for the cost investment and personal interest loss of reducing negative environmental externalities and improving positive environmental externalities; second, from the perspective of an externality double boundary, the ideas and principles of compensation pricing shall be put forward: the compensation standard should be determined from the vertical boundary of theoretical research and the horizontal boundary of practical application, combined with the government's ability to make financial payment.

Fertilizer Reduction Potential and Economic Benefits of Crop Production for Smallholder Farmers in Shaanxi Province

MI XiaoTian,SHI Lei,HE Gang,WANG ZhaoHui

Scientia Agricultura Sinica. 2021, 54(20):  4370-4384.  doi:10.3864/j.issn.0578-1752.2021.20.011

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【Objective】The material input of agriculture production is generally high for smallholder farmers, and the application rate of chemical fertilizers will change with the evolution of crop structure. Understanding the situation of nutrient input and economic benefits of smallholder to cereal crop, oil crop and cash crop could help guide scientific fertilization and improve economic benefits. 【Method】In 2018, a questionnaire survey was conducted on the production of major crops in Shaanxi Province. The cereal crop (wheat, maize, rice, millet, and potato), oil crop (canola and soybean), and cash crop (apple, tomato, and flue-cured tobacco) were considered as targeted crops, and a total of 1 709 questionnaires were obtained. Further, the fertilization status of smallholder, the potential of fertilizer reduction, and economic benefits according to yield level of different crops were evaluated. 【Result】For the production of wheat, maize, rice, millet, and potato, the mean yield was 4.6, 7.3, 8.3, 3.7, and 19.8 t·hm^-2, respectively; the mean nitrogen (N) fertilizer rate was 177, 247, 186, 255, and 209 kg N·hm^-2, respectively; the mean phosphate (P) fertilizer rate was 102, 103, 88, 142, and 125 kg P₂O₅·hm^-2; mean potassium (K) fertilizer rate was 37, 47, 64, 53, and 110 kg K₂O·hm^-2, respectively. For canola and soybean production, the mean yield was 2.4 and 2.7 t·hm^-2, N fertilizer rate was 156 and 99 kg N·hm^-2, P fertilizer rate was 80 and 63 kg P₂O₅·hm^-2, K fertilizer rate was 56 and 26 kg K₂O·hm^-2, respectively. For the production of apple, tomato, and tobacco, the mean yield was 23.8, 93.5, and 2.7 t·hm^-2, respectively; N fertilizer rate was 731, 471, and 118 kg N·hm^-2, P fertilizer rate was 482, 387, and 118 kg P₂O₅·hm^-2, K fertilizer rate was 535, 447, and 132 kg K₂O·hm^-2, respectively. N and P fertilizers were usually over-applied, while both over-application and under-application of K fertilizer coexisted. The potential of N, P, K fertilizer reduction ranges from 28% to 60%, 52% to 66%, and 11% to 51% for cereal crop, 33%, 37% and 46% for canola, respectively. However, the application rate of P and K fertilizer needed to increase by 11% and 28% for soybean production, respectively. The potential of N, P, and K fertilizer reduction ranged from 41% to 67%, 65% to 70%, and 49% to 64% for apple and tomato production, respectively. For tobacco production, the application rate of P fertilizer could decrease by 53%, while the application rate of N fertilizer needed to increase by 22% and 11% for farmers with medium and high-yielding, respectively. The source of N was mainly compound fertilizers and urea, and the sources of P and K were mainly compound fertilizers. For the way of chemical fertilizer application, most of N fertilizer was applied as basal fertilizer, and a small part was supplemented by topdressing, while almost all P and K fertilizers were applied as basal fertilizer. For cereal crop, oil crop, and cash crop, the cost of fertilizer application accounted for 31%-52%, 57%-59% and 48%-65% of total input, respectively, and the net economic benefit ranged from 0.44×10⁴to 1.63×10⁴, 0.75×10⁴to 0.84×10⁴, and 4.19×10⁴ to 15.05×10⁴yuan/hm², respectively. Because the net economic benefit of high-yielding in cash crop, e.g., apple and tomato, was higher than that of cereal crop and oil crop, smallholder farmers were more inclined to grow them. However, the substantial application of chemical fertilizer in cash crop production brought great environmental risks. 【Conclusion】The main target of fertilizer reduction and benefit improvement was smallholder with low and middle-yielding level. Compared with cereal crop and oil crop, the economic benefit of cash crop, especially for apple and tomato, was higher, while their potential of fertilizer reduction was also higher due to substantial application of chemical fertilizer. Notably, the result of the study also demonstrated that there was also insufficient application of fertilizer in crop production, e.g., soybean and tobacco. In conclusion, the fertilizer management of smallholder varied greatly. Improving the level of fertilizer management for smallholder was conducive to increasing both yield and economic benefits, and thus contributing to sustainable crop production.

Effects of Deep Plowing and Organic-Inorganic Fertilization on Soil Water and Nitrogen Leaching in Rice Field

WANG JinYu,CHENG WenLong,HUAI ShengChang,WU HongLiang,XING TingTing,YU WeiJia,WU Ji,LI Min,LU ChangAi

Scientia Agricultura Sinica. 2021, 54(20):  4385-4395.  doi:10.3864/j.issn.0578-1752.2021.20.012

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【Objective】Aimed at the problems of shallow soil plow layer, thickening of plow pan and soil hardening caused by perennial shallow ploughing and unreasonable fertilization in rice-wheat rotation area in the middle and lower reaches of Yangtze River in China,the effects of deep plowing (breaking part of plow pan) and fertilization on paddy field soil bulk density, soil compaction, soil water leaching and nitrogen leaching were studied to illuminate the response of nitrogen leaching to two tillage methods and three fertilization measures, and then provide theoretical basis for the construction of plow layer in the paddy soil. 【Method】 (1) Two tillage methods (rotary tillage 12 cm and deep plowing 20 cm) and three equal nitrogen fertilization treatments (single inorganic fertilizer treatment T1, returning straw with inorganic fertilizer treatment T2, organic manure with inorganic fertilizer treatment T3) were established in Shucheng County, Anhui Province in 2015. Soil water leaching and nitrogen leaching in rice season as well as soil bulk density and soil compaction were monitored dynamically in 2019-2020; (2) The soil-column experiment from paddy field was conducted to monitor. Water leaching from the treatment of deep plowing 30 cm in depth (total breaking of soil plow pan) was studied. 【Result】 (1) Field experiment results showed that the soil bulk density and soil compaction from the treatment of deep plowing 20 cm in depth were declined in rice season compared to those from the treatment of rotary tillage 12 cm in depth. Compared with the treatment of rotary tillage 12 cm in depth, the soil water leaching from the treatment of deep plowing 20 cm in depth increased by 7.4% in tillering stage, and there was no obvious change in soil water leaching after rice booting stage. From the whole rice growth period, the difference of soil water leaching between the treatment of deep plowing 20 cm in depth and the treatment of rotary tillage 12 cm in depth was not significant; (2) The results of soil-column experiment showed the soil water leaching from the treatment of deep plowing 30 cm in depth (total breaking of soil plow pan) increased significantly by 19.0% and 11.0% in flooding and 23.0% and 21.5% in non-flooding, respectively, compared with the treatment of rotary tillage 12 cm in depth and the treatment of deep plowing 20 cm in depth; (3) Nitrate nitrogen was dominant form of nitrogen in the soil water leaching. The concentration of nitrate nitrogen in soil water leaching from T3 treatment decreased significantly compared with that of T1 and T2 treatment after rice booting stage, but the difference of ammonium nitrogen concentration in soil water leaching from T1, T2 and T3 treatment were not significant; (4) From the whole growth period of rice, the difference of nitrogen leaching from the treatment of rotary tillage 12 cm in depth and the treatment of deep plowing 20 cm in depth was not significant, while the three treatments of fertilization had obvious difference on nitrogen leaching. Under the condition of deep plowing 20 cm in depth, the nitrogen leaching rates of T1, T2 and T3 treatment were 10.7, 11.7 and 9.1 kg N·hm^-2 respectively, and under the condition of rotary tillage 12 cm in depth, the nitrogen leaching rates of T1, T2 and T3 treatment were 9.83,11.21 and 8.58 kg N·hm^-2, respectively. T3 treatment decreased significantly nitrogen leaching compared to T1 and T2 treatment. 【Conclusion】 Deep plowing 20 cm in depth can improve soil physical structure, however, soil water leaching and nitrogen leaching are not significantly increased, and the combination of organic manure and inorganic fertilizer can significantly reduce nitrate nitrogen leaching. These results are of theoretical significance for the building of deep and fertile tillage layer in the clay paddy soil with high plow pan (such as red-yellow soil) in the middle and lower reaches of the Yangtze River through deep plowing measures, combined application of organic manure and inorganic fertilizer.

HORTICULTURE

Screening and Sequence Analysis of BAC Clone Contained PG Gene Controlling Clingstone/Freestone Characteristic of Peach

MENG JunRen,NIU Liang,DENG Li,PAN Lei,LU ZhenHua,CUI GuoChao,WANG ZhiQiang,ZENG WenFang

Scientia Agricultura Sinica. 2021, 54(20):  4396-4404.  doi:10.3864/j.issn.0578-1752.2021.20.013

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【Objective】Tandem repeats and large segment deletions of Polygalacturonase (PG) gene was related to the clingstone/freestone characteristic of peach (Prunus persica) fruit. In this study, the sequence character of F-M locus of clingstone peach was analyzed to provide a basis for the development of related molecular markers. 【Method】With a Bacterial Artificial Chromosome library of clingstone peach 87-7-1 constructed, the positive BAC clone contained F-M locus was screened from the BAC library by PCR analysis. The screened BAC clone was sequenced by single-molecule nanopore technology. Gene annotation and sequence alignment were performed by bioinformatics. 【Result】PCR primers were designed based on re-sequencing data of existing peach varieties, and PCR reactions were performed with all the BAC library clones. Amplification products of the sequences in upstream/downstream of F-M loci were corrected, and then the target clone 46-B-10 was obtained. Full-length sequencing showed that the fragment with the length of 111612 bp and GC content of 37.03% was inserted between upstream and downstream primers. The homologous region of 46-B-10 was determined by sequence alignment with the reference genome Prunus_persica_v2.0. Five genes (Prupe.4G261700, Prupe.4G261800, Prupe.4G261900, Prupe.4G262000, and Prupe.4G262500) in F-M locus were found in the BAC clone 46-B-10. In comparison, 87-7-1 with 34 kb sequence including four genes was discarded, and one of them was EndoPGF, which controlled peach freestone and was reported previously. 【Conclusion】Compared with the reference genome freestone variety ‘Lovell’, there was only EndoPGM (Prupe.4G261900) in F-M locus of clingstone peach individual 87-7-1, while EndoPGF (Prupe.4G262200) was discarded. In this study, the structural variation of F-M locus in clingstone peach was determined, which has laid an important foundation for the development of molecular markers for clingstone/freestone trait in peach.

The Influence of Rootstocks on the Growth and Aromatic Quality of Two Table Grape Varieties

SUN Lei,WANG XiaoYue,WANG HuiLing,YAN AiLing,ZHANG GuoJun,REN JianCheng,XU HaiYing

Scientia Agricultura Sinica. 2021, 54(20):  4405-4420.  doi:10.3864/j.issn.0578-1752.2021.20.014

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【Objective】The aim of this study was to investigate the aromatic characteristics of Muscat grape varieties, so as to provide a reference in selecting the optimal scion/rootstock combination and updating the vine management program. 【Method】Table grape varieties Ruiduxiangyu and Ruiduhongyu were grafted on five rootstock varieties (110R, 103P, SO4, 3309M, and 5BB) to form ten scion-rootstock combinations, while self-rooted vines were used as control. Head space solid-phase extraction method and gas chromatography mass spectrometry (GC-MS) was applied to extract the volatile and glycoside-bound compounds in the ripening berries, the compounds were identified by the library of National Institute of Standards and Technology version 2011 and retention index, and the standard curve was used to quantify/semi-quantify the compounds. And then, correlation analysis, principle component analysis and aromatic profile analysis were conducted to investigate differences among all the combinations. 【Result】A total of 56 volatile compounds and 33 glycoside-bound compounds were identified. The types of volatile compounds in these two varieties were not affected by 5BB. 2-Methyl-3-buten-2-ol, heptanal and 3-Methyl butanal were not detected in XY/3309M, whilst 1-hexanol and β-damascenone were not detected in HY/3309M. 1103P, 110R and SO4 could significantly increase the total content of volatile terpenoids in Ruiduxiangyu, while 5BB significantly reduced all kinds of volatile compounds. For Ruiduhongyu, these five rootstocks could increase the total content of free form terpenoid. The effects of five rootstocks on the glycoside-bound compounds in both varieties were insignificant. These two table grape varieties could be distinctly separated by principal components analysis, and 1-hexanol, geranic acid and linalool were the common biomarker compounds in all the rootstock of Ruiduhongyu. 110R, 1103P, SO4 and 3309M enhanced the citrus and floral flavor in Ruiduxiangyu, while 5BB stimulated the floral profiles in Ruiduhongyu. 【Conclusion】Therefore, the rootstock varieties of 110R, 1103P and SO4 were recommended to be used in the commercial production of Ruiduxiangyu, while 5BB was not suggested in practice. 5BB, 110R, 1103P, SO4, 5BB and 3309M were appropriate as the rootstock for Ruiduhongyu.

Fruit Quality in Storage, Storability and Peel Transcriptome Analysis of Rong’an Kumquat, Huapi Kumquat and Cuimi Kumquat

LIU Lian,TANG ZhiPeng,LI FeiFei,XIONG Jiang,LÜ BiWen,MA XiaoChuan,TANG ChaoLan,LI ZeHang,ZHOU Tie,SHENG Ling,LU XiaoPeng

Scientia Agricultura Sinica. 2021, 54(20):  4421-4433.  doi:10.3864/j.issn.0578-1752.2021.20.015

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【Objective】As a citrus fruit with edible peel, the fruit peel of kumquat affects not only the chewing texture but also fruit storage performance. With identical genetic background, Rong’an kumquat, Huapi kumquat and Cuimi kumquat exhibit significant differences in fruit peel. This study aimed to provide new insights and methods for kumquat fruit quality regulation and postharvest storage, through comparisons of three kumquats in fruit quality, storability, peel transcriptome analysis and effects of peel on postharvest characteristics. 【Method】Fruits of Rong’an, Huapi and Cuimi were harvested at commercial maturity stage and stored for 99 days at room temperature. During storage, fruit changes of water loss rate, soluble solid, acid, hardness and shear force were determined. Further, peel transcriptome analysis was performed for three kumquats. 【Result】The results showed that Huapi had the highest water loss rate and happened peel shrinking earliest among three cultivars. Water loss rate of Huapi was up to 38.6 % at 99 days after storage, significantly higher than 5.8% of Rong’an and 14.3% of Cuimi. Fruit soluble solid and acid in all three kumquats increased overall during storage. During storage, Cuimi exposed the highest soluble solid content always, while Huapi showed the lowest acid content since 22 days after storage. Cuimi and Huapi were with better inner quality during storage than Rong’an. Fruit structure of Rong’an changed tremendously during storage, and the fruit cells were damaged obviously at 44 days of storage. Fruit hardness and shear force of Huapi and Cuimi were significantly higher than those of Rong’an. Lignins (A₂₈₀·g^-1) in peels of Huapi and Cuimi were 1.41 and 1.31, respectively, which were all higher than 1.12 of Rong’an. Both Huapi and Cuimi showed more peel cellulose as well than Rong'an. Peel transcriptome analysis suggested that phenylpropaneiod biosynthesis pathway was up-regulated significantly in Huapi and Cuimi relative to Rong’an, while it changed scarcely between Huapi and Cuimi. Gene expression analysis showed that nine genes in lignin biosynthesis pathway expressed higher in Huapi and Cuimi. 【Conclusion】Three kumquats could be stored in a short time at room temperature, and being sold out in one month after harvest could be a good option for planter. In storage, Huapi kumquat lost commodity value quickly because of its fast water loss, while Cuimi kumquat performed well in both external and internal quality. Stronger fruit firmness and shear force as well as better storage performance of Cuimi were associated with the higher lignin and cellulose contents in fruit peel and closer cell arrangement in fruit. Lower peel lignin content in Rong'an kumquat caused by a weak phenylpropane biosynthesis was related closely to its poor peel toughness.

FOOD SCIENCE AND ENGINEERING

p-Coumaric Acid Promoted Wound Healing of Potato Tubers by Accelerating the Deposition of Suberin Poly Phenolic and Lignin at Wound Sites

LIANG Wei,ZHU YaTong,CHAI XiuWei,KONG Rui,LI BinShan,LI YongCai,BI Yang,DOV Prusky

Scientia Agricultura Sinica. 2021, 54(20):  4434-4445.  doi:10.3864/j.issn.0578-1752.2021.20.016

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【Objective】This study aimed to investigate the effect and related mechanism of p-CA treatment on the accumulation of suberin polyphenolic and lignin at wound sites of potato tubers. 【Method】The half cut potato tubers ‘cv. Atlantic’ were soaked with 0.5 mmol∙L ^-1p-CA for 10 min (using distilled water treated as control), which were stored at room temperature and protected from light for healing. The weight loss and the disease index of wounded tubers inoculated with Fusarium sulphureum were determined, the deposition of suberin polyphenolic and lignin at wounded sites were observed, and the activity of the key enzymes of phenylpropane metabolism and peroxidase as well as the content of phenylpropane metabolism products and H₂O₂ at wounded sites were measured. 【Result】The weight loss and the disease index of wounded tubers were significantly reduced by p-CA treatment, which were 38.46% and 43.18% lower than those of control on the 14 day of healing, respectively. p-CA treatment accelerated the deposition of suberin polyphenolic and lignin at wound sites of potato tubers, and the thickness of cell layers in treated tubers were 26.43% and 30.26% higher than that under control on the 14 day of healing, respectively. p-CA treatment also significantly increased the activities of phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase, 4-coumaryl coenzyme A ligase and cinnamyl alcohol dehydrogenase at wounde sites of tubers, with the enzyme activities of the treated tubers being 32.17%, 23.51%, 25.09% and 23.08% higher than the control on 7 day, respectively. p-CA treatment also promoted the synthesis of cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid and total phenolics, with the contents of cinnamic acid, p-coumaric acid, caffeic acid and total phenolics in the treated tubers on 14 day being 34.26%, 38.29%, 19.31% and 41.04% higher than that under control, respectively, and the contents of ferulic acid, sinapic acid in the treated tubers being 38.33% and 20.47% higher than that under control on 21 day, respectively. In addition, p-CA treatment promoted the accumulation of cinnamyl alcohol, coniferyl alcohol, sinapis alcohol and lignin at wound sites, with the contents of cinnamyl alcohol and lignin in the treated tubers being 36.93% and 31.66% higher than that under control on 14 day, respectively, and the contents of coniferyl alcohol and sinapis alcohol in the treated tubers were 41.43% and 34.05% higher than the control on 21 day, respectively. The p-CA treatment also increased the H₂O₂ content and peroxidase activity at the wound sites, with the H₂O₂ content and peroxidase activity of the treated tubers being 40.25% and 27.01% higher than that under control on 7 day, respectively. 【Conclusion】The p-CA treatment accelerated wound healing of potato tubers by activating phenylpropanoid metabolism, increasing H₂O₂ content and peroxidase activity, and promoting the accumulation of suberin polyphenolic and lignin in potato tuber wound sites.

Effects of Camellia Oil on the Properties of Myofibrillar Protein Gel

HAN KeYing,FENG Xiao,YANG YuLing,LI ShanShan,WEI SuMeng,CHEN YuMin

Scientia Agricultura Sinica. 2021, 54(20):  4446-4455.  doi:10.3864/j.issn.0578-1752.2021.20.017

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【Objective】The effects of camellia oil addition on the properties and the relationship between the properties and structure of myofibrillar protein (MP) gel were studied. The purpose of the research was to provide a theoretical basis for the application of camellia oil in chicken products. 【Method】The MP was extracted from chicken breast at 4℃. The MP heat-induced gels in different camellia oil addition were prepared. The changes of hardness, water holding capacity (WHC), moisture distribution of MP gel and rheological properties of MP were measured. Furthermore, the effects of camellia oil on the microstructure, secondary structure were determined by scanning electron microscopy (SEM) and Raman spectroscopy, respectively. Surface hydrophobicity and total sulfhydryl content were used to study the tertiary structure of MP in gels. Meanwhile, the differential scanning calorimetry (DSC) was applied to characterize the thermal properties of MP. Finally, the correlation between camellia oil addition, gel properties and MP structures were analyzed. 【Result】After 4% camellia oil addition, the hardness, WHC and the area fraction of bound water (PT₂₁) of MP gel increased and reached to maximum values of 116.60 g, 95.77% and 80.05%, respectively. However, those values decreased as camellia oil addition increased to 5%. For camellia oil content of 4%, the storage modulus (G') and elasticity of MP were the highest, while the surface roughness was the lowest. Meanwhile, MP gels showed more uniform, and compact network structure compared with other groups. It was also found that secondary structure of MP was significantly affected by camellia oil addition (0-5%). The α-helix content decreased from 39.94% to 26.07%, and the β-sheet content increased from 22.04% to 27.40% with increased camellia oil addition (P<0.05). The content of β-turn and random coil showed increasing trends. The S₀-ANS of MP reached the highest value, and the total sulfhydryl content was the lowest after 4% camellia oil addition. The results showed that the addition of camellia oil promoted the exposure of hydrophobic amino acid residues in protein molecules and the formation of disulfide bond, and changed the tertiary structure of MP in gels. The enhanced aggregation and cross-linking between MP molecules were due to the higher β-sheet content and hydrophobic interaction. The correlation analysis indicated that camellia oil addition was significantly correlated with the changes in the gel properties and protein structure (P<0.05). The results of DSC showed that camellia oil could interact with MP and reduce the denaturation enthalpy of MP. 【Conclusion】The camellia oil addition had a significant effect on the gel properties and structures of MP. The addition of camellia oil changed the microenvironment of MP and interacted with MP, which made the degeneration of MP easier. The secondary structure of MP was unfolded and the tertiary structure was affected after camellia oil addition, which improved the properties of MP gel. When camellia oil concentration was 4%, the properties and three-dimensional network structure of MP composite gel were the best.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Transcription Factor TEAD4 Regulates Early Embryonic Development in Pigs

ZHANG DanDan,XU TengTeng,GAO Di,QI Xin,NING Wei,RU ZhenYuan,ZHANG XiangDong,GUO TengLong,SHENTU LuYan,YU Tong,MA YangYang,LI YunSheng,ZHANG YunHai,CAO ZuBing

Scientia Agricultura Sinica. 2021, 54(20):  4456-4465.  doi:10.3864/j.issn.0578-1752.2021.20.018

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【Background】 TEA domain transcription factor 4 (TEAD4) is known to be a member of the TEAD family of transcription factors and plays a key role in determining the characteristics of the preimplantation embryo in rodents. In mouse embryos, it was found to be involved in regulating the genealogical differentiation of trophectoderm cells in preimplantation embryos by promoting Cdx2 expression. The absence of the TEAD4 gene in mouse embryos can lead to failure of mouse blastocyst formation. However, the role of TEAD4 in early porcine embryonic development is still unclear. 【Objective】This study aimed to preliminarily elucidate the effect of TEAD4 on early porcine embryonic development, in order to lay the theoretical foundation for further exploring the molecular mechanisms of transcription factors on early porcine embryonic development. 【Method】In this study, the bioinformatics analysis of the porcine TEAD4 gene was performed by using web-based tools, including analysis of the porcine TEAD4 gene sequence, comparison of homology between pigs, human and mice, and comparison of the evolutionary relationship of TEAD4 between different species. The role of TEAD4 in early embryonic development in pigs was then tested. The mRNA expression level of TEAD4 gene in porcine oocytes and the early embryos was firstly detected by fluorescence quantitative PCR. and then, siRNA targeting TEAD4 was designed and injected into mature oocytes by microinjection technique to reduce the level of endogenous TEAD4 gene in the oocyte cytoplasm, and to determine that TEAD4 siRNA acts only on TEAD4 gene, with a view to determining the role of TEAD4 gene in early porcine embryonic development. 【Result】Sequence analysis showed that the porcine TEAD4 gene contained 11 exons and localized on chromosome 5, with spanning 37.188 kb, 1 473 bp in full mRNA length, and 1305 bp in full coding region, which encoded 434 amino acids. Homology analysis with human and mouse revealed that TEAD4 was highly conserved in different species and had the closest affinity on pig and cow. The results of fluorescence quantitative PCR showed that TEAD4 mRNA was expressed in both porcine oocytes and early embryos; compared with GV-stage oocytes, the expression of TEAD4 mRNA was lowest in MII-stage oocytes and remained low until the 4-cell stage, but reached the highest expression in the 8-cell stage, and then gradually decreased in the morula and blastocyst stages. Microinjection of siRNA targeting TEAD4 revealed that TEAD4 siRNA only acted on the endogenous TEAD4 gene in oocytes, but not on TEAD1 and TEAD3, and compared with the control and negative control siRNA groups, the injection of TEAD4 siRNA significantly reduced TEAD4 mRNA expression at the 8-cell and morula embryo periods. When TEAD4 gene expression was knocked down, observation of the developmental efficiency of porcine orphan activation and in vitro fertilization embryos showed that the developmental efficiency of TEAD4 siRNA knockdown group from 8-cell to blastocyst stage was significantly reduced compared to the control and negative control siRNA groups. 【Conclusion】 The results of this study indicated that the TEAD4 gene was highly conserved across species, with the closest affinity on pigs and bovine, and that TEDA4 might be involved in regulating the development of early porcine embryos..

Alternative Splicing of TNNT3 and Its Effect on the Differentiation of MuSCs in Goat

CHEN Yuan,CAI He,LI Li,WANG LinJie,ZHONG Tao,ZHANG HongPing

Scientia Agricultura Sinica. 2021, 54(20):  4466-4477.  doi:10.3864/j.issn.0578-1752.2021.20.019

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【Objective】As a member of the troponin (Tn) family, TNNT3 (Fast Skeletal Troponin T3) involves skeletal muscle contraction, growth, development, and even meat characteristics of domestic animals. This study initially aimed to identify the alternative splicing of goat (Capra hirus) TNNT3. 【Method】Based on goat TNNT3 (NM_001314210. 1) and cattle TNNT3 (XM_010821200) mRNA sequence from NCBI (National Center for Biotechnology Information), the primers were designed by using the Primer Premier 6.0 software, subsequently, TNNT3 was amplified from skeletal muscles of embryo Jianyang Bigear Goat. The obtained TNNT3 sequences were then bioinformatically analyzed by using ORF Finder, EditSeq, DNAMAN, ClustalW, and MEGA_X_10.1.8. Furthermore, the levels of TNNT3 isoforms were quantified by using real-time fluorescence quantitative PCR (RT-qPCR) and semi-quantitative PCR in longissimus dorsi (LD) muscle, semimembranosus (SM) muscle, heart, liver, spleen, lung, and kidney, at seven stages (E75, E90, E105, B3, B45, B150, and B300), respectively. Additionally, the coding ability of transcript TNNT3_3 in vitro and its effect on the differentiation of goat skeletal muscle satellite cells (MuSCs) were explored. 【Result】① A total of five isoforms (named TNNT3_1-5) of the TNNT3 gene were identified in pooled RNA extracted from goat muscles, and the complete coding sequence (CDS) sequence mainly contained 18 exons. ② Nucleotide sequence and amino acid sequence of the goat TNNT3 gene were highly consistent with sheep, cattle, pig, and other mammals, but low with that of fish and reptiles, indicating the high evolutionary conservation of the TNNT3 gene in mammals. ③ The TNNT3 mRNA was presented in all seven detected tissues but highly enriched in LD and SM muscles (P < 0.01), followed by cardiac muscle and lung. Furthermore, the levels of TNNT3 mRNA in SM muscles were higher than that in LD muscles in prenatal goats (P < 0.05), while the verse results were presented postnatal (P < 0.05). ④ The conserved exon 9-11 (138 bp) of goat TNNT3 was repeated in the transcript TNNT3_3. TNNT3_3 was amplified, and it was found out that which could encode protein, and the protein size was basically the same as expected (37 kDa) in TnT transcriptional translation system in vitro. Moreover, the transfection of TNNT3_3 into goat MuSCs induced mRNA levels of myogenic differentiation marker genes, including Myomaker, MyoG, and MyH4, comparing with the control (P < 0.01). 【Conclusion】Five isoforms with complete CDS region of the TNNT3 gene were obtained in goat muscles. The sequence and expression of TNNT3 were highly conserved in mammals and enriched in muscles, indicating that potentially TNNT3 gene functions critically in muscle growth and development.

Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody

LI MinXue,LI JianNan,ZHOU Hong,XIAO Ning,LIN HuiXing,MA Zhe,FAN HongJie

Scientia Agricultura Sinica. 2021, 54(20):  4478-4486.  doi:10.3864/j.issn.0578-1752.2021.20.020

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【Objective】 Lawsonia intracellularis (L. intracellularis) is an enteric pathogenic bacteria that causes porcine proliferative enteropathy (PPE), which mainly shows the decline of animal welfare and causes serious economic losses to the world swine industries. The objective of this study was to prepare monoclonal antibodies against SodC of L. intracellularis, and to establish an immunoperoxidase monolayer assay (IPMA) method for detecting L. intracellularis base the monoclonal antibody, while test its application in clinical practice, so as to provide a scientific and effective means for the diagnosis of L. intracellularis. 【Method】In this study, the commercial live attenuated L. intracellularis vaccine was selected as target strain. The sodc gene was amplified by PCR and cloned into the prokaryotic expression vector pGex-6p-1. The recombinant plasmid pGex-6p-1-sodc was confirmed to be constructed successfully and induced expression of recombinant SodC protein. The reactivity of the recombinant protein was analyzed by Western Blot. The primary antibody was a mouse anti-GST labeled antibody. BALB/c mice aged 4-6 weeks were immunized with purified SodC protein, and hybridoma cells were screened by conventional cell fusion, limited dilution and indirect ELISA, then ascites were prepared. It was confirmed that two monoclonal antibodies had good specificity through indirect immunofluorescence (IFA). Using the monoclonal antibody as the primary antibody, a method of IPMA for detecting L. intracellularis was developed, and the specificity, sensitivity and repeatability of the method were evaluated. The optimized IPMA method was used to detect ileal tissue samples from pig farms in Jiangsu Province and to evaluate the clinical value of the method. 【Result】After purification, the concentration of SodC protein was higher, and it specifically bound to the antibody against GST tag, indicating that the protein had good regenicity. After three times of subcloning, two strains positive hybrid tumor cells were screened, named 1D6 and 1F7, respectively. The titers of two monoclonal antibodies were both reached 1﹕204 000 by ELISA. The subclass identification results of antibodies showed the subclass of 1D6 was IgA,and subclass of 1F7 was IgG3. The result IFA showed that 1D6 and 1F7 had specific reaction with L. intracellularis, but did not cross-react with S. Cholerasuis, PEDV and TGEV. The ascites of the two monoclonal antibodies were both 1﹕1 024 000 by ELISA; IFA confirmed that the two monoclonal antibodies had good specificity. The optimized IPMA reaction conditions showed that when the dilution ratio of the primary antibody was 1:800 for 45 min, and the dilution ratio of the secondary antibody was 1﹕2 500 for 1h, the established IPMA exhibited the best performance. The specificity and sensitivity tests showed that S. Cholerasuis, PEDV, TGEV, PCV2 and PRV were all negative, and the minimum detection limit was 10³L.intracellularis. The optimized IPMA method was used to detect the ileum tissue samples from pig farms in the surrounding areas of Jiangsu Province. A total of 92 positive samples were detected from 146 samples of ileal tissues. The positive rates of 3 different pig farms were 65.6%, 68.1% and 53.7%, respectively, and the overall positive rate was 63.0%. 82 positive samples were detected by PCR method. and the positive coincidence rate of the two methods was 94.6%. These results indicated that this method had clinical value. 【Conclusion】The monoclonal antibodies against the recombinant SodC protein were successfully prepared, and the IPMA method for L. intracellularis was established with good specificity and sensitivity, and the clinical samples were tested. In summary, these results further proved that the IPMA had certain clinical value, and provided an effective technical means for the isolation and identification of L. intracellularis in the laboratory, localization in infected cells, epidemiological investigation and quarantine.