Scientia Agricultura Sinica

PSORA: A Strategy Based on High-Throughput Sequence for Analysis of T-DNA Insertion Sites

MA XueMeng,YU ChengMin,SAI XiaoLing,LIU Zhen,SANG HaiYang,CUI BaiMing

Scientia Agricultura Sinica. 2022, 55(15): 2875-2882. doi:10.3864/j.issn.0578-1752.2022.15.001

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【Objective】The purpose of this study was to establish a simple and efficient approach for identifying all T-DNA insertion sites. 【Method】A T-DNA insertion sites analysis approach based on high-throughput sequence technologies was developed, called PSORA: Parallel sequencing of one round amplicons. The process involves high-throughput amplicon sequencing of a round of thermal asymmetric PCR (TAIL-PCR) and bioinformatics analysis of T-DNA insertion sites, which reduces concerns about the specificity of TAIL-PCR. In PSORA, only two primers are required, a degenerate primer and a T-DNA specific primer. A 6-nt Barcode was designed at the 5’ end of the specific primers for labeling different transgenic events. All five transgenic events (L1, L6, L9, L15 and L19) of tobacco used in this study were produced via Agrobacterium mediated transformation with plasmids pBI121. In addition, the results of PSORA are confirmed by standard PCR. 【Result】The T-DNA insertion sites of five transgenic events were analyzed by PSORA. The results showed that L6 contained two insertion sites (36 316 bp on NW_015801367 and 42 202 bp on NW_015950898), the lines of L9, L15 and L19 each contained one insertion site (The insertion site of L9 was located at 235 969 bp on NW_015943682. The insertion site of L15 was located at 60 529 bp on NW_015802951 and the insertion site of L19 was located at 12 188 bp on NW_015863435), but the insertion site of L1 could not be detected. PCR was performed to validate the results from bioinformatics analysis, transgenic events with different insertion sites were used as negative controls for each other, and the wild type (WT) was used as a blank control. The results showed that specific amplification consistent with expectations was obtained in each transgenic event. The effectiveness of PSORA was successfully confirmed. 【Conclusion】PSORA is an effective strategy to analyze T-DNA insertion sites. PSORA can parse the comprehensive molecular characteristics of all T-DNA insertion events simultaneously, making it simpler and faster than the traditional methods of genome walking.

QTL Analysis for Growth Period and Panicle-Related Traits in Foxtail Millet

GUO ShuQing,SONG Hui,CHAI ShaoHua,GUO Yan,SHI Xing,DU LiHong,XING Lu,XIE HuiFang,ZHANG Yang,LI Long,FENG BaiLi,LIU JinRong,YANG Pu

Scientia Agricultura Sinica. 2022, 55(15): 2883-2898. doi:10.3864/j.issn.0578-1752.2022.15.002

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【Objective】Growth period and panicle-related traits are the main factors affecting foxtail millet yield and adaptability. To analyze the molecular genetic mechanisms of the growth period and panicle-related traits, it is necessary to map the related genes. 【Method】A recombinant inbred line (RIL) population of foxtail millet consisting of 400 lines derived from a cross between the elite varieties, Yugu18 and Jigu19, were used in this study. Phenotype surveys were carried out across four independent environments in 2018—2019 to study the days from emergence to heading and heading to maturity, the growth period, panicle length, panicle diameter, and single panicle weight. Based on a high-density genetic map that spanned 2 196 cM of the foxtail millet genome with an average of 1.68 cM/bin, composed of 1 304 bin markers, QTL for growth period and panicle-related traits were mapped using composite interval mapping (CIM), and the candidate genes of the confidence interval were predicted. 【Result】The growth period and panicle-related traits of RIL population exhibited continuous distribution with apparent transgressive segregation in the 4 environments, which accorded with the genetic characteristics of typical quantitative traits and were suitable for QTL genetic analysis. Correlation analysis showed that the days from emergence to heading were significantly positively correlated with the growth period, and negatively correlated with those heading to maturity. A positive correlation between panicle length and diameter was also observed. A total of 88 QTL for the growth period and panicle-related traits were detected on chromosomes 1, 3, 5, 6, 8 and 9, respectively. Among them, 45 QTL were significantly associated with the days from emergence to heading and explained 1.61%-27.60% of the phenotypic variance; seven QTL were significantly associated with the days from heading to maturity and explained 2.65%-12.14% of the phenotypic variance; 20 QTL were significantly associated with the growth period and explained 1.98%-16.97% of the phenotypic variance; nine QTL was significantly associated with the panicle length and explained 3.51%-11.65% of the phenotypic variance; five QTL were significantly associated with the panicle diameter and explained 3.74%-8.34% of the phenotypic variance; two QTL was significantly associated with the single panicle weight and explained 5.16%-5.20% of the phenotypic variance. A total of 12 major QTL were detected in this study, of which five major QTL, including qEHD-9-1, qEHD-9-2, qHMD-9-2, qGRP-9-2 and qPL-5-1, were repeatedly detected in at least two environments and BLUP value. The genomic regions of qEHD-9-1, qHMD-9-1, qGRP-9-1 and qPL-9-1 overlapped on chromosome 9, the genomic regions of qEHD-9-2, qHMD-9-3, qGRP-9-2 and qPL-9-3 overlapped on chromosome 9, and the genomic regions of qPL-5-1 overlapped with qPD-5-1 on chromosome 5. Five candidate genes related to the growth period and panicle-related traits were identified from the confidence interval of the 3 QTL clusters, of which two candidate genes played an important role both in the growth period and panicle-related traits.【Conclusion】A total of 88 QTL for the growth period and panicle-related traits were detected, and 12 were major QTL, of which 5 major QTL were repeatedly detected in multiple environments by clustering distribution. Five candidate genes related to the growth period and panicle-related traits were identified via gene annotation.

Effects of Arbuscular Mycorrhizal Fungi Inoculant Diversity on Yield, Phosphorus and Potassium Uptake of Maize in Acidic Soil

ZHANG ChenXi, TIAN MingHui, YANG Shuo, DU JiaQi, HE TangQing, QIU YunPeng, ZHANG XueLin

Scientia Agricultura Sinica. 2022, 55(15): 2899-2910. doi:10.3864/j.issn.0578-1752.2022.15.003

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【Objective】The aim of this study was to clarify the effects of different arbuscular mycorrhizae fungi (AMF) inoculants and their diversity on maize grains yield and the absorption and accumulation of phosphorus (P) and potassium (K) in acidic soil, so as to provide a theoretical basis for the application of farmland microbial fertilizer and the improvement of crop nutrient uptake. 【Method】Four different AMF species (Funneliformis mosseae, A; Glomus tortuosum, B; Glomus versiforme, C; Funneliformis geosporum, D) were selected as experimental materials in 2019 and 2020, and a pot experiment with sixteen treatments was established during maize growth periods, including CK (non-inoculating AMF species), inoculating single AMF treatment, two AMF mixed treatments, three AMF mixed treatments, and four AMF mixed treatments. The maize grain yield, plant biomass and their P and K accumulation were analyzed. 【Result】Compared with CK, the presence of each type of AMF species could increase maize grain yield and their P and K accumulation. Compared with CK, the presence of A, B, C and D increased maize grain yield by 57.9%, 26.9%, 40.5% and 32.9%, respectively, increased the maize grain P accumulation by 77.8%, 48.2%, 26.2% and 75.8%, respectively and increased maize grain K accumulation by 85.5%, 62.1%, 59.0% and 63.5%, respectively. With AMF inoculant diversities increasing, the average values of maize grain yield showed an increasing tendency, with four AMF mixed treatments>three AMF mixed treatments>two AMF mixed treatments>single AMF treatments, and the average grain yield of four AMF mixed treatments was 12.3% higher than that of the single AMF treatments. The tendency of grain P accumulation among the AMF diversity treatments was two AMF mixed treatments>four AMF mixed treatments>three AMF mixed treatments>single AMF treatments, and the average grain P accumulation for two AMF mixed treatments was 23.2% higher than that of single AMF treatments. However, there was no significant difference of grain K accumulation among AMF diversity treatments. 【Conclusion】In acidic soil, the inoculation of different AMF species could significantly increase maize grain yield and their P and K accumulation, and the effects of Funneliformis mosseae was best on improving maize grain yield and their P and K accumulation among four AMF species. The increase of AMF inoculant diversities could significantly improve maize grain yield and their P accumulation, while the four mixed AMF showed a better effect on grain yield, and on grain nutrient uptake for two mixed AMF. All of these indicated that, according to the purposes of high grain yield or high grain quality in production, the suitable types of AMF species should be selected to inoculate their host, and the mix of different types of AMF species should matched reasonably.

Effects of Intertillage Time and Depth on Photosynthetic Characteristics and Yield Formation of Soybean

YUAN Cheng, ZHANG MingCong, WANG MengXue, HUANG BingLin, XIN MingQiang, YIN XiaoGang, HU GuoHua, ZHANG YuXian

Scientia Agricultura Sinica. 2022, 55(15): 2911-2926. doi:10.3864/j.issn.0578-1752.2022.15.004

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【Objective】Aiming at the low temperature in early spring and poor moisture retentionability of main soybean producing areas in the northern part of Northeast China, the effects of different intertillage times and depths on soil temperature and humidity, photosynthetic characteristics and yield of soybean field under maize-soybean rotation mode were explored to provide a strong support for the stable improvement of soybean yield. 【Method】The experiment was carried out in Heshan farm, Heilongjiang province from 2019 to 2020. The plot experiment method was adopted, and Heihe 43, the main local cultivator, was used as the test material. Four different intertillage treatments were set up: conventional earthing-up (T1), advanced earthing-up (T2), conventional subsoiling (T3), and advanced subsoiling (T4). The effects of intertillage time and depth on soil temperature and humidity, plant leaf area index and height, gas exchange parameters, accumulation and distribution of photosynthetic products and yield of soybean were studied.【Result】(1) On the basis of the same intertillage depth, compared with T1, soil temperature and humidity in flowering stage (R2 stage) under T2 increased by 5.88%-6.54% and 3.57%-4.03% (P<0.05), respectively, and leaf area index, plant height and SPAD values in seed-filling stage (R6 stage) were increased by 9.48%-16.86%, 5.40%-10.57% and 2.39%-6.81% (P<0.05), respectivley. Compared with T3, T4 significantly increased leaf area index, plant height, net photosynthetic rate (P_n), grain dry matter accumulation and soybean yield at R6 stage. (2) Compared with T1, T3 increased soil temperature and humidity in R2 stage by 4.14%-6.42%, 10.08%-13.19% and plant height in R6 stage by 7.43%-8.29% (P<0.05), respectively. After pod-setting stage (R5 stage), dry matter accumulation and assimilation contribution rate increased by 49.75% and 32.95% (P<0.05), respectively. Compared with T2, T4 significantly increased soil temperature, leaf area index at R6 stage, net photosynthetic rate (P_n), dry matter accumulation after R5 stage, assimilation contribution rate and yield, among which the yield increased by 5.03%-6.02% (P<0.05). (3) Compared with T1, T4 increased soil temperature and humidity by 11.68%-17.15% and 4.70%-8.66% in R2 stage (P<0.05), respectively. Leaf area index, plant height and SPAD in R6 stage were increased by 12.64%-27.42%, 11.67%-13.50% and 5.43%-6.87% (P<0.05), respectively. T2, T3 and T4 increased gas exchange parameters and yield in R6 stage, and net photosynthetic rate (P_n) under T4 treatment increased by 14.25%-29.68%, and yield increased by 10.69%-18.71% (P<0.05).【Conclusion】T4 could improve soil temperature and humidity, increase gas exchange parameters, promote plant net photosynthate accumulation, and delay leaf senescence, finally increase soybean yield, which was suitable for the promotion and application of dry farm areas in the north of Northeast China.

A Salt Stress Tolerance Effect Study in Peanut Based on Peanut//Sorghum Intercropping System

SHI XiaoLong, GUO Pei, REN JingYao, ZHANG He, DONG QiQi, ZHAO XinHua, ZHOU YuFei, ZHANG Zheng, WAN ShuBo, YU HaiQiu

Scientia Agricultura Sinica. 2022, 55(15): 2927-2937. doi:10.3864/j.issn.0578-1752.2022.15.005

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【Objective】The main objectives of this study were to investigate the response of peanut to salt stress under peanut//sorghum intercropping, and hope to provide new insights on develop stress cultivation. 【Method】 In this study, peanut cultivar “Huayu 25” and sorghum cultivar “Liaoza 15”, with characteristics of salt tolerance and a high yield potential, were selected as experimental materials to carry out the field planting box experiment for two consecutive years. Sole-cropped peanut (SP) and intercropped peanut (IP) experiments were then performed under normal (N) and salt stress (S) soil conditions, respectively. The experiment was comprised of four treatments: sole-cropped peanut under normal condition (N-SP), intercropped peanut under normal condition (N-IP), sole cropped peanut under salt stress condition (S-SP), and intercropped peanut under salt stress condition (S-IP). Therefore, the salt tolerance index (STI), relative interaction index (RII), Na⁺/K⁺, and rhizosphere nutrient of peanut were investigated in the present study to evaluate the response of peanut to salt stress under different planting patterns.【Result】In the peanut//sorghum intercropping system, both RII of peanut were negative. However, the negative RII was decreased significantly and the STI was increased significantly under S-IP with salt stress, especially after continuously being planted for two years. Of these, the negative RII of S-IP decreased by 66.78% in 2019 than that of in 2018, and the negative RII under S-IP decreased by 88.76% than that under N-IP. Furthermore, the STI under S-IP increased by 27.68% than that under S-SP in both 2018 and 2019. Peanut//sorghum intercropping has been found to change the overall root distribution and architecture by favoring the development of different types of roots, and also affected rhizosphere nutrients of peanut, the rhizosphere soil nutrient content of N-IP and S-IP increased by an average of 6.19% and 3.73% than N-SP and S-SP, respectively. Under salt stress, the content of soil potassium increased significantly compared with normal soil conditions, this might be the initial defensive response utilized by plants to maintain Na⁺/K⁺ homeostasis in rhizosphere soil, which regulated Na⁺/K⁺ homeostasis in peanut by influencing the Na⁺ and K⁺ selective absorption and transportation. Compared with S-SP, the leaf Na⁺/K⁺ ratio of S-IP decreased by 20.63%, the leaf salinity hazard coefficient (LSHC) decreased by 53.95%, so the photosynthetic potential and light energy conversion efficiency were significantly improved too. Ultimately the dry matter accumulation capacity and yield potential were improved, in which the yield potential under S-IP had the most obvious increase, and the yield under S-IP increased by 17.95% in 2019 compared with 2018.【Conclusion】The continuous peanut intercropped with sorghum under salt stress could be an effective technique to alleviate peanut negative interactions, which significantly improved STI and alleviate salt stress of peanut by improving soil nutrient status and regulating peanut Na⁺/K⁺ homeostasis, which ultimately maintained the dry matter accumulation capacity and increased yield potential.

Quantitative Proteomic Analysis of Spirotetramat Inhibiting Hatching of Frankliniella occidentalis Eggs

ZHOU GuiYing,YANG XiaoMin,TENG ZiWen,SUN LiJuan,ZHENG ChangYing

Scientia Agricultura Sinica. 2022, 55(15): 2938-2948. doi:10.3864/j.issn.0578-1752.2022.15.006

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【Objective】Previous studies showed that the 0 h old eggs of Frankliniella occidentalis after 24 h treated with spirotetramat resulted in the cracking of eggs shell and inhibited the hatching of eggs. The objective of this study is to investigate the causes of morphological and structural changes of 0 h old eggs of F. occidentalis after 24 h treated with spirotetramat, and to find the key proteins and reveal the molecular mechanism of spirotetramat inhibiting the hatching of 0 h old eggs of F. occidentalis. 【Method】The 0 h old eggs of F. occidentalis were treated with 14.42 mg·L^-1 spirotetramat for 24 h, and the Label-free quantitative proteomics technique was used to analyze differentially expressed proteins between the spirotetramat-treated group and the control group. Gene Ontology (GO) enrichment analysis and KEGG pathway analysis were used for functional annotation and pathway analysis of the differentially expressed proteins, and the expression of differential expressed proteins was verified by parallel reaction monitoring (PRM).【Result】A total of 204 proteins were differentially expressed after treatment with spirotetramat, of which 124 proteins were up-regulated and 80 proteins were down-regulated. Bioinformatics analysis showed that the up-regulated differentially expressed proteins were mainly involved in substance synthesis and metabolic pathways, while the down-regulated differentially expressed proteins were mainly participated in the defense response pathway to external stimuli. In addition, the results of PRM were consistent with the proteomics results. The results showed that the protein expression levels of GTP cyclohydrolase 1-1, GTP cyclohydrolase 1-2, glutathione S-transferase-like and fatty acyl-CoA reductase were significantly up-regulated, while the expression levels of endocuticle structural glycoprotein SgAbd-2-like, superoxide dismutase [Cu-Zn]-like and O-acetyl transferase in 0 h old eggs of F. occidentalis after 24 h treated with spirotetramat were significantly down-regulated, especially endocuticle structure glycoprotein SgAbd-2-like.【Conclusion】The above proteins may play multiple functions in the hatching process of F. occidentalis eggs, the endocuticle structure glycoprotein SgAbd-2-like is related to insect epidermal differentiation and other life activities, it was speculated that the expression of this protein was inhibited by spirotetramat, which resulted in egg shell rupture of F. occidentalis 0 h old eggs, the endocuticle structure glycoprotein SgAbd-2-like may play an important role in the hatching process of F. occidentalis 0 h old eggs.

Commercialization Status and Existing Problems of RNA Biopesticides

GUAN RuoBing,LI HaiChao,MIAO XueXia

Scientia Agricultura Sinica. 2022, 55(15): 2949-2960. doi:10.3864/j.issn.0578-1752.2022.15.007

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RNA biopesticides use the principle of RNA interference (RNAi) to inhibit the expression of important genes in target organisms, causing the developmental retardation or death of harmful organisms, thereby achieving the purpose of pest control. The technology does not alter the genome of pests and cause adverse effects on the ecosystem. RNA biopesticides are called “the third revolution in the history of pesticides” because they have the advantages of precision, high efficiency, green and pollution-free, etc. In recent years, with the approval of Bayer’s insect-resistant transgenic maize MON87411 which expressing insect dsRNA, major traditional agrochemical companies have invested a lot of manpower and material resources in layout and product development. In addition, it has also attracted the attention of the capital market, and large numbers of companies based on RNAi technology for pest control have emerged, which has greatly accelerated the industrialization of RNA biopesticides. With the rapid development of RNA biopesticides, it will be bound to change the global pesticide market pattern, which is undoubtedly a new challenge. Although the R&D program in this field started early and the starting point is relatively high in our country, most of the research mainly focuses on basic theories, and the application development is relatively weak, which has lagged far behind the international counterparts. Compared with traditional pesticides, RNA biopesticides have their own unique features in both mechanism and application development. It is urgent to improve the corresponding laws and regulations to supervise and guide production, promote the rapid development of RNA biopesticides in our country, and reduce the risk of international pesticide giants forming a technological monopoly in this field. Based on this, this paper systematically summarized the current domestic and foreign R&D status, commercialization, and future development trends of RNA biopesticides, as well as the regulations and policies related to RNA biopesticides in Europe, the United States and other countries. In addition, the paper also pointed out some urgent problems in the progress of R&D and industrialization of RNA biopesticides, hoping to provide a useful reference for the development and supervision of RNA biopesticides in China.

Research on Spatial Distribution of Soil Texture in Southern Ningxia Based on Machine Learning

SHEN Zhe,ZHANG RenLian,LONG HuaiYu,XU AiGuo

Scientia Agricultura Sinica. 2022, 55(15): 2961-2972. doi:10.3864/j.issn.0578-1752.2022.15.008

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【Objective】Based on historical soil data, this paper studied the spatial variability of soil texture and its relationship with environmental factors in southern Ningxia by using machine learning.【Method】Classification and regression tree (CART), random forest (RF) and traditional statistical methods were used to explore the main environmental factors that affected the soil texture types and predict the spatial distribution of soil texture types in southern Ningxia, based on 428 soil profiles from the second soil survey in the 1980s, combined with topographic factors, soil types, and normalized vegetation index. The accuracy of the models were verified by the validating set of soil profiles and the soil samples in Haiyuan County, Ningxia.【Result】(1)The accuracy rates of RF and CART on the soil texture type of the verification set of soil profiles were 62.36% and 55.29%, respectively; the area under the receiver operating characteristic (ROC) curve of them (area under roc curve, AUC) were 0.7515 and 0.6933, respectively; the accuracy rates of them on soil samples in Haiyuan County were 54.10% and 48.36%, respectively; the AUC of them were 0.6599 and 0.5981 respectively. (2) Soil type (ST) was the most important predictor variable, followed by elevation (Ele). The higher elevation was, the heavier the soil texture was. The effects of wind exposition index (WEI) and slope (Slo) on soil texture were lower. (3)The results predicted by two methods showed a spatial distribution trend that the soil texture was heavy in the southern area but light in the northern area of southern Ningxia.【Conclusion】The prediction accuracy of RF for soil texture type in southern Ningxia was higher than CART. Making full use of historical data, combined with field sampling, could meet the accuracy requirements of digital mapping. In the loess region, soil types and elevation were the environmental factors which had strong correlation with spatial variation of soil texture.

Quantitative Study on Effective Accumulated Temperature and Dry Matter and Nitrogen Accumulation of Summer Maize Under Different Nitrogen Supply Levels

CHEN Yang,XU MengZe,WANG YuHong,BAI YouLu,LU YanLi,WANG Lei

Scientia Agricultura Sinica. 2022, 55(15): 2973-2987. doi:10.3864/j.issn.0578-1752.2022.15.009

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【Objective】This paper explored the dynamic prediction model and characteristic parameters of dry matter and nitrogen accumulation in summer maize with different nitrogen supply levels based on effective accumulated temperature, in order to provide a theoretical basis for using effective accumulated temperature to predict summer maize dry matter and nitrogen accumulation.【Method】This study was based on a two-year field experiment in Langfang, Hebei Province (2019-2020), using Zhengdan 958 as the test material, and using the normalization method to fit the dry matter and nitrogen accumulation of summer maize with different nitrogen supply levels through model screening. Based on the normalized Gompertz model of effective accumulated temperature after sowing, and using the growth rate curve and its characteristic parameters, the dry matter and nitrogen accumulation characteristics of summer maize were quantitatively analyzed.【Result】(1) Under the experimental conditions, when the amount of phosphorus and potassium fertilizer was appropriate, the maximum dry matter and nitrogen accumulation of summer maize continued to increase with the increase of nitrogen application rate. (2) The normalized Gompertz model of summer maize dry matter and nitrogen accumulation established with effective accumulated temperature as the independent variable had the good biological significance. The coefficients of determination of the equation were 0.9962-0.9988 and 0.9887-0.9922, respectively. Using the second-year data for model verification, the correlation coefficients of the simulated and measured values were 0.9933-0.9959 and 0.9830-0.9923, and the standardized root mean square errors were 6.64%-16.86% and 7.31%-12.68%, respectively. The prediction effect was good. (3) The growth rate of dry matter and nitrogen accumulation of summer maize at different nitrogen supply levels all showed a “single peak curve”, and its change was closely related to the nitrogen supply level. The performance between treatments was: under the condition of moderate fertilization, the growth rate curve had the characteristics of fast rising and falling, and the growth rate curve of weight loss treatment had the characteristics of slow rising and falling. (4) The effective accumulated temperature ranges of dry matter and nitrogen accumulation during the rapid increase period of summer maize after sowing were 709.35-1 722.54 and 482.50-1 507.61 ℃·d, respectively, and the effective accumulated temperature required for the maximum rate showed that nitrogen accumulation (995.05 ℃·d) was less than dry matter accumulation (1 215.94 ℃·d). Nitrogen supply level obviously affected the accumulation of dry matter and nitrogen in summer maize to enter the accumulation temperature required for the rapid increase period, the accumulated temperature required for the slow increase period, the accumulated temperature required for the maximum increase rate, the maximum increase rate, and the average increase rate during the rapid increase period. Compared with nitrogen fertilizer treatment, the effective accumulated temperature required for summer maize to enter each critical period was significantly reduced, and the growth rate during the critical period increased significantly.【Conclusion】The normalized Gompertz model could not only simulate and predict the dynamic changes of summer maize dry matter and nitrogen accumulation with effective accumulated temperature with different nitrogen supply levels, but also clarify the quantitative relationship between effective accumulated temperature and dry matter and nitrogen accumulation. The Gompertz model based on effective accumulated temperature could be used to predict crop growth and optimal fertilization period, and had strong application value..

Effects of Partial Substitution of Chemical Fertilizer by Organic Fertilizer on Ponkan Growth and Quality as well as Soil Properties

WAN LianJie,HE Man,LI JunJie,TIAN Yang,ZHANG Ji,ZHENG YongQiang,LÜ Qiang,XIE RangJin,MA YanYan,DENG Lie,YI ShiLai

Scientia Agricultura Sinica. 2022, 55(15): 2988-3001. doi:10.3864/j.issn.0578-1752.2022.15.010

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【Objective】Through systematical research focused on the effects of different organic fertilizer substitutions on growth, yield and quality, physicochemical properties of soil and orchard environment of ponkan under equal nutrient, it could be provided the suitable amount of organic fertilizer substitution for the scientific application of organic fertilizer and theoretical basis for enlarging high quality of citrus.【Method】The 15-year-old Taiwan ponkan grafted on Poncirus trifoliata (L.) was used as experimental materials. Six different treatments were performed, including no fertilization (CK), single chemical fertilizer (FP), substitution 10% of nitrogen (N), 25% of phosphorus (P), 18% of potassium (K) (T1), substitution 15% of N, 38% of P, 27% of K (T2), substitution 20% of N, 51% of P, 36% of K (T3), substitution 30% of N, 76% of P, 54% of K (T4). The nitrogen, phosphorus and potassium nutrient content of treatment except CK was consistent with research into the effects of different organic fertilizer substitution on the accumulation of citrus branches, leaves and fruits of dry matter in that year, fruit yield and quality, physicochemical properties of soil, carbon emissions and nitrogen leaching in 2019 and 2020.【Result】(1) The two-year’s research showed similar trends in growth indicators, such as spring and autumn leaf, fruit and flower of dry matter, with the growth of organic fertilizer substitution showing a trend of first rise and then decrease. On the whole, T1 and T2 treatment were beneficial to accumulate ponkan dry matters of leaf, fruit and flower. In 2019 and 2020, the yield of each organic fertilizer substitution treatment increased by 7.2% to 26.4% and 2.0% to 36.9%, respectively. (2) The soluble solids treated by organic fertilizer substitution treatment increased significantly by 7.4% to 9.8% compared with FP in 2020. Among other quality, T2 and T3 were better on the whole. (3) Each organic fertilizer substitution treatment of soil physicochemical properties promoted compared under FP in 2019 and 2020. The soil pH and organic matter treated by organic fertilizer will be significantly increased by 4.6% to 12.6% and 16.4% to 25.1%, respectively. (4) Compared with FP, each organic fertilizer substitution treatment of soil carbon flux was higher by 20.1%, while which of nitrous nitrogen was lower by 13.9%. With the increase of organic substitution ratio, the migration of nitrous nitrogen to deep soil was lower. (5) Through the analysis of the principal component and the comprehensive score of research index on ponkan’s growth, yield and quality, physicochemical properties of soil and environment, the score was sorted as T3>T2>T4>T1>FP>CK.【Conclusion】T3 (organic fertilizer replacement 20% of N, 51% of P, 36% of K) was the best under this research, and this treatment was not only beneficial to the growth and development of citrus trees, but also conducive to the formation of good physical, chemical characteristics of the soil and fruit high-quality yield, to a certain extent, to reduce the risk of greenhouse gas emission and soil nitrogen leaching.

Analysis of Genetic Diversity of 79 Cultivars Based on SSR Fluorescence Markers for Peach

WANG LuWei,SHEN ZhiJun,LI HeHuan,PAN Lei,NIU Liang,CUI GuoChao,ZENG WenFang,WANG ZhiQiang,LU ZhenHua

Scientia Agricultura Sinica. 2022, 55(15): 3002-3017. doi:10.3864/j.issn.0578-1752.2022.15.011

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【Objective】The aim of this study was to perform genotyping and genetic diversity analysis of 79 germplasms taken from National Fruit Germplasm Nanjing Peach Resource Nursery by using SSR fluorescent-labeled capillary electrophoresis technology, and to screen primers with high polymorphism, which could be used for identification of peach varieties and genetic relationship analysis and also be used for the establishment of molecular marker-assisted selection system.【Method】De novo sequencing was performed on the female parent Zhongyou No. 4, and SSR markers were developed in the whole genome with a physical distance of 1 Mb as the unit. 79 peach varieties in Nanjing were used as materials for PCR amplification, and the amplified products were detected by polyacrylamide gel electrophoresis and screened for markers with clear target bands and abundant polymorphisms. The 5' end of the screened markers were fluorescently modified, and the PCR products were sequenced on the ABI3730XL sequencer to realize the rescreening of the markers. Genmapper 4.0 software was used to count the sequencing results, and Data Formater 2.1 software converted the bp data obtained by statistics into the data format required by Power Marker v3.25. The selected primers were analyzed for polymorphism, and the fluorescent SSR primers with polymorphism information content (PIC) greater than 0.45 were selected as the core primers of 79 germplasm materials. Using Nei's as a parameter, the UPDM clustering method in NTSYSpc 2.1 software was used to analyze the genetic diversity among cultivars. The Structure v2.3.4 software based on Bayesian model was used to analyze the population genetic structure of 79 accessions. 【Result】Based on 79 germplasms, the SSR markers covering the whole gene were screened by polyacrylamide gel electrophoresis, and 207 pairs of primers with good polymorphism were screened out. The 207 pairs of primers were re-screened by SSR fluorescent-labeled capillary electrophoresis technology, and 26 pairs of core primers were finally screened, of which 5 pairs of core primers could completely distinguish 79 varieties. 26 pairs of SSR core primers amplified a total of 174 polymorphic genotypes in 79 peach germplasms, and each pair of primers were amplified 4-13 genotypes, with an average of 6.69 genotypes amplified per pair of primers. The polymorphism information content PIC of each pair of primers was above 0.45. Based on the locus information amplified by 207 pairs of SSR primers in 79 accessions, the genetic relationship map and population genetic structure map of 79 accessions were constructed. 207 pairs of primers divided 79 accessions into 7 groups based on genetic distance and 2 groups based on population structure. The genetic diversity of 79 cultivars was high, and the clustering results of some cultivars were consistent with the pedigree. 【Conclusion】The cluster analysis map and population genetic structure map of 79 materials were constructed, revealing the genetic relationship of 79 materials to a certain extent. The overall results showed that due to the complex genetic background of peach, the cultivars according to a single characteristic or determine the relationship between cultivars could not be classified. The screened 26 pairs of core primers could be used for genome-wide identification of linked traits, identification of peach germplasm resources, identification and protection of new varieties, and construction of molecular marker-assisted breeding systems.

Evaluation of Brown Rot Resistance in Peach Based on Genetic Resources Conserved in National Germplasm Repository of Peach in Nanjing

SHEN ZhiJun, TIAN Yu, CAI ZhiXiang, XU ZiYuan, YAN Juan, SUN Meng, MA RuiJuan, YU MingLiang

Scientia Agricultura Sinica. 2022, 55(15): 3018-3028. doi:10.3864/j.issn.0578-1752.2022.15.012

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【Objective】 The brown rot is one of the most serious diseases in peach throughout the world and usually causes huge economic losses during the peach harvest and storage stages. The systematic evaluation on brown rot resistance of peach germplasm resources and screening of resistant resources were performed for developing new peach cultivars with high resistance to brown rot, and finally solving the problem in peach cultivation.【Method】 The commercially matured peaches with 8.0-8.5 maturity degree from National Germplasm Repository of Peach in Nanjing were inoculated by Monilinia spore during 2018-2021. Altogether, 616 accessions were evaluated by uninjured inoculation, and 505 accessions by injured inoculation. The percentage growth of infected fruits per day (PGIF, %) and lesion diameter growth rate (LDGR, cm∙d^-1) were used to evaluate brown rot resistance, and then a nine-grade evaluation method was established based on the average value of PGIF and LDGR with 0.5 standard deviation, respectively. The brown rot resistance was compared among different germplasm types, fruit types and origins, and the correlation between brown rot resistance and other characteristics were also investigated. 【Result】 PGIF was (11.22±5.96)% with the CV value of 35.48 for the samples with uninjured inoculation. Based on the nine-grade resistance evaluation method by PGIF, the accession with Grade 1 (PGIF<0.80%) was not available, ten accessions with Grade 2 (0.80%-3.78%), 134 accessions with Grade 3 (3.78%-6.76%), 157 accessions with Grade 4 (6.76%-9.73%), 122 accessions with Grade 5 (9.73%-12.71%), 73 accessions with Grade 6 (12.71%-15.68%), 60 accessions with Grade 7 (15.68%-18.66%), 21 accessions with Grade 8 (18.66%-21.64%), and 39 accessions with Grade 9 (>21.64%). LDGR was (1.71±0.21) cm∙d^-1 with the CV value of 0.18, and there were 11 accessions with Grade1 (LDGR<0.98 cm∙d^-1), 28 accessions with Grade 2 (0.98-1.19 cm∙d^-1), 72 accessions with Grade 3 (1.19-1.40 cm∙d^-1), 109 accessions with Grade 4 (1.40-1.61 cm∙d^-1), 103 accessions with Grade 5 (1.61-1.83 cm∙d^-1), 82 accessions with Grade 6 (1.83-2.04 cm∙d^-1), 45 accessions with Grade 7 (2.04-2.25 cm∙d^-1), 29 accessions with Grade 8 (2.25-2.46 cm∙d^-1), and 26 accessions with Grade 9 (>2.46 cm∙d^-1) according to the nine-grade resistance evaluation method by LDGR. The linear regression of PGIF (y) to LDGR (x) was y=6.2073x, with a low R²value of 0.1839. The comparison of brown rot resistance among different groups showed that the wild peach resources originated from China had more resistance to brown rot relative to those from other areas, and the wild resources would be very valuable for peach brown rot resistance breeding in the near future. PGIF and LDGR were significantly negatively correlated with both fruit firmness with exocarp (-0.234) and without exocarp (-0.240), and significantly positively with fruit size (0.427) and degree of coloring (0.319), but the correlation coefficient was relatively low. 【Conclusion】 A nine-grade evaluation system for peach brown rot resistance was established based on percentage growth of infected fruits per day (%) and lesion diameter growth rate (cm∙d^-1), respectively. In addition, 10 peach germplasm resources with high resistance to infestation by peach brown rot and 11 with high anti-expansion capacity against peach brown rot were screened in this study.

Effects of Very Fast Chilling on Flavor Quality in Chilled Lamb

YAN TongJing,ZHANG DeQuan,LI Xin,LIU Huan,FANG Fei,LIU ShanShan,WANG Su,HOU ChengLi

Scientia Agricultura Sinica. 2022, 55(15): 3029-3041. doi:10.3864/j.issn.0578-1752.2022.15.013

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【Objective】The objective of this study was to clarify the effect of very fast chilling on flavor quality of chilled lamb, so as to provide a theoretical and experimental basis, to some extent, for developing very fast chilling technology. 【Method】Lamb Silverside muscles were obtained from hot carcasses at 1 h postmortem, and were chilled under conventional chilling (chilling rate 1.94℃∙h^-1) and very fast chilling (chilling rate 15.10℃∙h^-1), respectively. The contents of nucleotides, free amino acids and volatile compounds were determined during storage. The taste active value and odor activity value were calculated to identify the key volatile compounds in chilled lamb. 【Result】5'-GMP, Alanine and Glutamic acid were identified as the key taste contributors in chilled lamb based on the taste activity value. 13 volatile compounds were identified as key flavor contributors of chilled lamb according to the odor activity value, including 8 aldehydes, 4 alcohols and 1 furan. Specifically, the 8 aldehydes included pentanal, (E)-2-octenal, nonanal, heptanal, (E, E)-2,4-nonadienal, (E)-2-nonenal, octanal, hexanal, the 4 alcohols included hexanol, (E)-2-octene-1-ol, octanol, 1-octen-3-ol, and the furan was 2-pentyl-furan. With the prolongation of storage time, 5'-AMP, 5'-IMP and 5'-GMP were gradually degraded into hypoxanthine and inosine, and sweet amino acids (serine), bitter amino acids (valine, methionine, leucine, tyrosine, phenylalanine, lysine, arginine) and volatile compounds (including 1-octen-3-ol, hexanal, octanal) increased significantly, which changed the flavor quality of chilled lamb in conventional chilling group. The degradation of 5'-AMP, 5'-IMP, 5'-GMP and the formation of free amino acids (including glutamic acid, glycine, proline, valine) and volatile compounds (including 1-octene-3-ol, hexanal, octanal, nonanal, heptanal, hexanol, 2-pentyl-furan) were delayed in very fast chilling group. This change maintained the freshness and reduced negative effects of bitter amino acids, aldehydes and alcohols on the flavor quality of chilled lamb. The result of the cluster analysis showed that flavor quality of chilled lamb at 72 h postmortem in very fast chilling group was similar to that of chilled lamb at 6 h postmortem in conventional chilling group. 【Conclusion】Very fast chilling could delay the changes of volatile compounds and taste compounds, therefore it maintained the flavor quality of chilled lamb in the state of pre-rigor.

Evaluating the Application Effect of Single-Step Genomic Selection in Pig Populations

ZHOU Jun,LIN Qing,SHAO BaoQuan,REN DuanYang,LI JiaQi,ZHANG Zhe,ZHANG Hao

Scientia Agricultura Sinica. 2022, 55(15): 3042-3049. doi:10.3864/j.issn.0578-1752.2022.15.014

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【Background】The contribution of genetic breeding is the highest in improving the efficiency of animal production. Through breeding, the animal husbandry enterprises can improve production efficiency and obtain maximum economic benefits. Genome selection has been widely used in plant and animal breeding. Genomic selection can estimate breeding values (EBV) by using high density markers covering the whole genome. Compared with pedigree information, the average relationship between individuals obtained by using these markers is more accurate, so that breeding values can be more accurately estimated, and the individuals can be selected. In practical breeding program, all individuals are not genotyped, especially for pigs, whose economic values are not high enough, hence the application of genomic selection is limited in pig breeding. The single-step genomic best linear unbiased prediction (ssGBLUP) can utilize both pedigree and genotypes information, allowing part of individuals are genotyped, thus greatly reducing genotyping costs while maintaining high prediction accuracy. At present, many studies have shown that the use of genomic selection in pig breeding can improve the accuracy of prediction, but in actual breeding, the breeding cost is also an important issue in livestock enterprises to consider. Therefore, how to implement breeding program economically and effectively is of great research value.【Objective】The effect of one-step genome selection on the population evaluation of Duroc was investigated, so as to provide the basis for genome selection breeding program.【Method】 In this study, three important economic traits of duroc pig born from 2009 to 2018 in a pig farm in Fujian province were studied. The accuracy of BLUP, GBLUP, and ssGBLUP was compared in calculating the estimated breeding value on reproductive and growth traits of Duroc pigs. The impacts of genotyped individuals with different proportions in the reference population on the ssGBLUP prediction abilities were explored, and the influence of different chip density on GBLUP prediction abilities was also studied. 【Result】 (1) The heritability of the age at 100 kg, the backfat, and eye muscle area was 0.257±0.038, 0.250±0.039 and 0.399±0.040, respectively; (2) Compared with BLUP, the accuracy of ssGBLUP was improved by 14.7%-51.1%; compared with GBLUP, the accuracy increased by 13.4%-45.7%; (3) When 10%-30% of individuals was genotyped, the prediction accuracy of ssGBLUP could exceed that of BLUP; the prediction accuracy reached a plateau when 40%-60% of individuals genotyped. 【Conclusion】Based on the above results, it was concluded that: (1) Compared with BLUP, ssGBLUP could improve the accuracy and reliability of EBV for each trait; compared with the GBLUP, ssGBLUP was slightly lower than the GBLUP with only pedigree information of those ungenotyped, but ssGBLUP performed better than the GBLUP method after the addition of phenotypes of the ungenotyped individuals. (2) As the proportion of genotyped individuals in the reference population increased, no matter which selection method was used to determine genotyped individuals (random selection and selection the key individuals), the prediction ability of ssGBLUP was gradually improved. The results showed that the ssGBLUP could improve the prediction ability of individual breeding value even if only a proportion of individuals was genotyped when the breeding budget was limited.

CTNNB1 Regulates the Function of Porcine Ovarian Granulosa Cells

LI LiYing,HE YingTing,ZHONG YuYi,ZHOU XiaoFeng,ZHANG Hao,YUAN XiaoLong,LI JiaQi,CHEN ZanMou

Scientia Agricultura Sinica. 2022, 55(15): 3050-3061. doi:10.3864/j.issn.0578-1752.2022.15.015

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【Objective】The ovarian granulosa cells proliferation and differentiation are important to the initiation of primordial follicle growth. The excessive of ovarian granulosa cells apoptosis is one of the main reasons for follicular atresia. Therefore, the functions of ovarian granulosa cells are very important for follicular growth, development, ovulation, hormone secretion and so on. The objective of this study was to investigate the effects of CTNNB1 on the function of granulosa cells proliferation, apoptosis and steroid hormone secretion during porcine follicle development, so as to provide a reference for the molecular regulation mechanism of porcine follicular development. 【Method】In this study, the expressions of CTNNB1 in porcine ovary, muscle, brain and other 9 tissues were detected by RNA extraction and qRT-PCR. Then, qRT-PCR and Western blot were used to explore the expression of CTNNB1 in different stages of porcine ovarian tissue, as well as granulosa cells of small (≤3 mm), medium (3-6 mm) and large (≥6 mm) follicles. The eukaryotic expression vector and siRNA of CTNNB1 were constructed and transfected into porcine ovarian granulosa cells. The effects of CTNNB1 on the proliferation, apoptosis and steroid hormone secretion of granulosa cells were detected by EdU, annexin V-FITC/PI double staining assay, flow cytometry, and ELISA. Finally, qRT-PCR was also applied to explore the effect of CTNNB1 on the expression of genes related to steroid hormone synthesis. 【Result】The expression of CTNNB1 mRNA in ovary was higher than that in other tissues. The expression of CTNNB1 mRNA in ovaries of in-sexually-mature sows was significantly higher than that of pre-sexually-mature and post-sexually-mature sows. The expression of CTNNB1 in the follicles was gradually increased with the development of follicles, and the expression of CTNNB1 in granulosa cells of follicles was also increased with the development of follicles. In addition, the expression of CTNNB1 in granulosa cells as well as medium and large follicles was extremely higher than that in small follicles. CTNNB1 could promote the proliferation of granulosa cells and inhibit the apoptosis of granulosa cells. Moreover, CTNNB1 could also up-regulate the transcription level of CYP1A1 and HSD17B7, and down-regulate the transcription level of CYP11A1, ESR1, ESR2, FSHR, LHR and NR5A1, which were related to steroid hormone synthesis, thereby promoting the secretion of estrogen and suppressing the secretion of androgen and progesterone in granulosa cells. 【Conclusion】The above results confirmed that CTNNB1 regulated the function of granulosa cells by stimulating its proliferation and secretion of estrogen, and restraining its apoptosis and secretion of androgen and progesterone, thereby promoting the growth and development of follicles.

Preparation of Monoclonal Antibodies Against the p30 Protein of African Swine Fever Virus and Its Mapping of Linear Epitopes

WEI Tian,WANG ChengYu,WANG FengJie,LI ZhongPeng,ZHANG FangYu,ZHANG ShouFeng,HU RongLiang,LÜ LiLiang,WANG YongZhi

Scientia Agricultura Sinica. 2022, 55(15): 3062-3070. doi:10.3864/j.issn.0578-1752.2022.15.016

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【Objective】In this study, monoclonal antibodies (MAbs) against the p30 protein of African swine fever virus (ASFV) were prepared and the linear epitopes on p30 was analyzed, which could lay the foundation for ASFV and its antibody detection as well as the study of p30 protein structure and function.【Method】BALB/c female mice aged 6 to 8 weeks were immunized with prokaryotic expression and purified recombinant p30 protein. The mice were immunized once every two weeks, with three times in total. First immunization was done with emulsification of antigen and equal volume Freund’s complete adjuvant, then, the mice were immunized with emulsification of antigen and equal volume of Freund’s incomplete adjuvant for the second and third immunization. After three immunizations, the tail was cut off and the blood was collected, and the serum antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titer were selected for enhanced immunization. Three days later, the mice spleen lymphocytes and SP2/0 myeloma cells were fused with PEG at a ratio of 4﹕1. The positive hybridoma cells were screened by indirect ELISA by using the recombinant p30 protein as coated antigen. and the MAbs which could secrete antibodies steadily were cloned and purified by limited dilution method. ASFV was inoculated into porcine alveolar macrophages, and the indirect immunofluorescence assay (IFA) was performed with MAbs as primary antibody and rabbit anti-rat HRP-IgG as secondary antibody. The ASFV-infected and uninfected cells were precipitated and transferred to the nitrocellulose membrane by SDS-PAGE. p30 MAbs were obtained by Western blotting analysis of MAbs and identified positive by IFA, which was used as primary antibody and rabbit anti-rat HRP-IgG as secondary antibody. Primers were designed to amplify two truncated genes p30ab and p30bc, p30ab stands for truncated amino acid residues at position 86-153 and p30bc stands for truncated amino acid residues at position 120-153. The recombinant protein GST-p30ab and recombinant protein GST-p30bc were obtained by partial overlapping truncated p30 protein expression in prokaryotes. GST-p30ab and GST-p30bc fusion proteins were used as coated antigens, and the epitopes of p30 protein were preliminarily identified by indirect ELISA with 5 MAbs as primary antibodies, with rabbit anti-rat HRP-IgG as secondary antibodies. 【Result】The purified recombinant protein was used as the coated antigen, and 25 hybridoma cell lines were screened by indirect ELISA, which could secrete anti-recombinant p30 protein. IFA results showed that 5 MAbs (8F4, 1D3, 1H2, 6C3 and 8E11) were positive for ASFV-infected cells. Western blotting results showed that all 5 strains of MAbs could react positively with ASFV-infected cells and negatively with uninfected cells. The recombinant p30 protein GST-p30ab was expressed in soluble and inclusion body forms, and GST-p30bc was expressed in inclusion body form. The truncated fusion proteins of the two groups were used as the coated antigens. Indirect ELISA showed that MAbs 8F4, 1H2 and 6C3 could effectively bind to the two recombinant proteins that means MAbs 8F4, 1H2 and 6C3 recognized the epitope amino acid 120-153; MAbs 8E11 and 1D3 could only bind to GST-p30ab protein, which meant they recognized the epitope amino acid 86-119. 【Conclusion】In this study, the recombinant p30 protein with amino acid truncated at position 86-153 was expressed in soluble form. Five MAbs were prepared and two p30 protein epitopes were located. Combined with ELISA and IFA, it could be formed a very reliable method for the detection of ASFV and its antibody.

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