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01 July 2018, Volume 51 Issue 13

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Scientia Agricultura Sinica. 2018, 51(13):  0. 

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Comprehensive Evaluation of Main Agronomic Traits of Millet Resources Under Different Light and Temperature Conditions

JIA XiaoPing, YUAN XiLei, LI JianFeng, ZHANG Bo, ZHANG XiaoMei, GUO XiuPu, CHEN ChunYan

Scientia Agricultura Sinica. 2018, 51(13):  2429-2441.  doi:10.3864/j.issn.0578-1752.2018.13.001

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【Objective】To uncover the characteristics of photo-thermo reaction of spring millet and summer millet, screen out the suitable traits for foxtail millet photo-thermo sensitivity evaluation, ten main agronomic traits including heading date, plant height, number of leaves, panicle length, panicle diameter, branch number per panicle, grain number per branch, panicle weight, grain weight per panicle and 1000-grain weight of 100 spring millet materials, 60 summer millet materials were investigated in two locations of Hainan and Jilin with different photo-thermo conditions.【Method】Based on the survey data of ten traits from 100 spring millet materials, 60 summer millet materials in two locations for two consecutive years, three methods including variance analysis, regression analysis and path analysis between ten traits and comprehensive evaluation index D value of photo-thermo sensitivity, photo-thermo relative sensitivity comparison analysis of ten traits were performed to evaluate the photo-thermo sensitivity of ten traits of spring millets and summer millets.【Result】Variance analysis showed that ten traits of both spring millets and summer millets were significant difference between two different photo-thermo conditions (p＜0.01), indicating the omnibearing effect of photo-thermo condition on spring millet and summer millet. The investigation values of heading date, plant height, number of leaves, panicle length, panicle diameter, branch number per panicle, grain number per branch, panicle weight, grain weight per panicle of both spring millets and summer millets in Jilin location were higher than that in Hainan location. On the contrary, the investigation value of 1000-grain weight under Jilin photo-thermo condition was lower than that under Hainan photo-thermo condition. Comprehensive evaluation index D value of photo-thermo sensitivity was constructed by principal component analysis, based on which regression analysis and path analysis were performed between ten traits and D value. The results showed that panicle weight, panicle length, grain weight per panicle and grain number per branch of spring millets had relatively high direct effect on D value in two investigation years, indicating a higher sensitivity of these traits to photo-thermo reaction. For summer millets, there was profound difference between two years, only panicle weight and panicle length had significant effect on D value, were sensitive to photo-thermo reaction in two years. By comparing the relative photo-thermo sensitivity of ten traits, it was found that panicle weight, grain weight per panicle, heading date, panicle length and branch number per panicle of spring millets were more sensitive to photo-thermo reaction than other traits in two years. Meanwhile, panicle weight, panicle length, heading date, branch number per panicle and number of leaves of summer millets were more sensitive to photo-thermo reaction than other traits in two years. Combining the results by D value regression analysis, path analysis, photo-thermo relative sensitivity analysis for two years, and the research results of rice, we could conclude that for spring millets, panicle weight, grain weight per panicle, panicle length, branch number per panicle heading date showed strong photo-thermo sensitivity, following them were number of leaves and plant height, panicle diameter and 1000-grain weight showed weak photo-thermo sensitivity. The photo-thermo sensitivity of grain number per branch was uncertain. For summer millets, panicle weight, panicle length, heading date, number of leaves and branch number per panicle showed strong photo-thermo sensitivity, panicle diameter, grain number per branch and 1000-grain weight showed weak photo-thermo sensitivity. The photo-thermo sensitivity of grain weight per panicle and plant height was uncertain. Panicle weight, panicle length and heading date were the mutual traits of spring millets and summer millets that showed strong sensitivity to photo-thermo reaction.【Conclusion】Spring millets and summer millets were both significantly affected by photo-thermo conditions. Panicle weight, panicle length and heading date of spring millets and summer millets were more sensitive to photo-thermo reaction, which were suitable index traits for photo-thermo sensitivity evaluation of foxtail millet. Number of leaves was suitable for photo-thermo sensitivity evaluation of summer millets. Plant height was not suitable for photo-thermo sensitivity evaluation of foxtail millet.

Identification and Expression Analysis of CRK Gene Family in Upland Cotton

ZHANG ZhongQi, WANG Jiao, JIN Wei, GE DongDong, LIU Kang, Lü FenNi, SUN Jing

Scientia Agricultura Sinica. 2018, 51(13):  2442-2461.  doi:10.3864/j.issn.0578-1752.2018.13.002

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【Objective】 The cysteine-rich receptor kinase (CRK) is one of the largest family of receptor-like kinases in plants, and plays important roles in plant growth and development, hormone signal transduction and stress tolerance. So far, few cotton CRK genes have been reported. In silico identification, bioinformatics and expression analysis of CRK family genes in upland cotton on whole genome level lays the foundation for in-depth study and utilization of CRK family genes in cotton. 【Method】 The conserved stress-antifung domain sequence downloaded from the Pfam database was used as query sequence to search the Gossypium hirsutum (cv. TM-1) genome database to identify cotton CRKs by using BLASTp program; the theoretical isoelectric point and molecular weight, signal peptide, transmembrane domain, subcellular localization of cotton CRK proteins was predicted applying Compute pI/Mw, SignalP, TMHMM Server V2.0, WoLF POSRT online program respectively; amino acid sequence alignment of CRK proteins in cotton and Arabidopsis thaliana was performed using ClustalX1.8 software. phylogenetic relationships of cotton and Arabidopsis CRK proteins were analyzed with MEGA5.0; the chromosome location, gene structure and conserved domain were visualized with TBtools. The promoter sequences of cotton CRK genes were In silico analyzed by searching PlantCARE database; phosphorylation sites were predicted with PlantPhos. RNA-Seq data were download the from the NCBI database, TPM values were calculated using transcriptome quantification tool Kallisto, the heatmap of CRK gene expression was drawn with online tools Morpheus. 【Result】 There are 70 CRK genes in upland cotton genome distributed on 14 chromosomes, 52 genes (74.3% of the total) are intensively distributed in clusters on A6/D6, A9/D9 and A10/D10 chromosomes characterized by collinear relationships between A/D chromosomes. These CRK genes encode proteins containing 302-901 amino acids, 58 proteins (82.9%) have a transmembrane domain, mainly located in the chloroplasts, plasmalemma and extracellular. Phosphorylation site prediction results showed that cotton and Arabidopsis CRK share 5 consensus phosphorylation sites, including three serine phosphorylation motifs and two threonine phosphorylation motifs. The promoter regions of 65 cotton CRK genes (account for 92.9%) contain at least one stress hormone response element, and 69 (98.6%) genes contain at least one biotic or abiotic stress response element. RNA-Seq data analyses showed that the tissue expression patterns of CRKs could be divided into three types, and that the expression of some CRK genes were altered in response to salt, drought, cold, heat stress and inoculation with Verticillium dahliae Kleb. GhCRK25 was predominantly expressed in roots, stems, leaves, and ovules, but barely accumulated in fibers. ABA, GA3, SA, PEG-6000, NaCl, and Verticillium dahliae Vd991 can stimulate rapid up-regulation of GhCRK25 expression. GhCRK25-silenced cottonby using virus induced gene silencing technology (VIGS) showed increased susceptibility to Verticillium dahliae Vd991. 【Conclusion】 There are 70 members of CRK family gene in the upland cotton genome. They have conserved gene structure and functional domain, diverse tissue expression characteristics; most of cotton CRK genes are responsive to hormone and stress stimulus.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Change of Atmospheric Environment Leads to Deterioration of Rice Quality

JING LiQuan, HU ShaoWu, MU HaiRong, WANG YunXia, YANG LianXin

Scientia Agricultura Sinica. 2018, 51(13):  2462-2475.  doi:10.3864/j.issn.0578-1752.2018.13.003

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Climate change will change the growth environment of crops, thereby affecting crop yield and quality. The effects of climate change on the yield formation of rice, one of the most important grain crops, had been reported extensively, but there were few studies on rice quality, which bore equal importance as yield in terms of rice production safety. After a brief introduction of the experimental platform, this paper summarized the research progress of the impact of climate change on rice quality. Quality traits were classified into processing, appearance, cooking/eating, nutritional and feeding quality. Climate change included elevated atmospheric CO₂ concentration, elevated tropospheric O₃ concentration, higher temperature etc. This paper focused on the interactions between atmospheric composition change and high temperature on rice quality. Previous studies showed many uncertainties about the impact of climate change on rice quality, but some important trends had also been found. Unfortunately, most of these trends indicated unfavorable changes in rice quality. Rice growing in high CO₂ concentration, high O₃ concentration or high temperature environment exhibited an increase in grain chalkiness and a higher percentage of broken grains during milling process. The concentrations of protein and several micronutrients in rice grains decreased with high CO₂ concentration, but the palatability was improved; both the eating quality of rice grain and feeding quality of rice straw showed a trend of deterioration when plants were growing under ozone stress. At present, the understandings in this area were obtained mostly from the impact of single climatic factor, but the interaction between CO₂ and temperature or O₃ had been observed in a few studies. In addition, the responses of rice quality traits to climate change might also be affected by fumigation methods, genotypes and fertilizer application. In future, the experimental platform of different scales should be employed to verify the existing trends; more efforts should devote to evaluate the interactions between climate change factors and other factors, and to reveal the mechanisms of these interactions; and all related researches should aim at the successful development of rice production technology that could truly adapt to future climate change.

Effects of Combined Application of Manure and Chemical Fertilizers on Loss of Gaseous Nitrogen and Yield of Summer Maize

YANG QingLong, LIU Peng, DONG ShuTing, ZHANG JiWang, ZHAO Bin, LI RongFa, REN Hao, REN Han, HAN XiangFei

Scientia Agricultura Sinica. 2018, 51(13):  2476-2488.  doi:10.3864/j.issn.0578-1752.2018.13.004

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【Objective】 This study monitored emission regularity and loss of NH₃ and N₂O under different treatments in the summer maize fields on the long-term experiment platform, to explore effective ways to reduce gaseous nitrogen losses in summer maize field in the Huanghuaihai region, in order to provide a theoretical basis for improving the yield of summer maize and fertilizer use efficiency. 【Method】 The experiment was conducted with lysimeters in 2016-2017. The maize cultivar of Zhengdan 958 (ZD958) was used as experimented material. The venting method was used to monitor NH₃ volatilization, and the static box-gas chromatography method was used to monitor losses of N₂O under four fertilization modes, including organic manure (M₁), organic manure plus chemical N fertilizer (1/2 N from organic manure, 1/2 from urea; U₂M₂), Urea (U₁) and no N fertilizer (CK). The content of nitrogen in M₁, U₁ and U₂M₂ treatment were equal. 【Result】 Loss of gaseous nitrogen in maize field was dominated by NH₃ volatilization, accounting for 88.55%-96.42% of the total nitrogen gaseous loss and N₂O emissions was less. NH₃ volatilization was the highest under U₁ treatment, with an average of 38.19 kg·hm^-2 of 2016 and 2017; the lowest treatment with M₁ was 19.10 kg·hm^-2, indicating that single application of organic fertilizer or organic-inorganic combination application could significantly reduce NH₃ volatilization. N₂O emission was highest under M₁ treatment, which reached an average of 1.65 kg·hm^-2, and then 77.42% and 34.15% higher than those of U₁ and U₂M₂ treatment, respectively. Nitrogen use efficiency of U₂M₂ treatment was the highest, with an average of 58.20%; compared with M₁ and U₁ treatment, which increased 32.15% and 15.13%, respectively. The results showed that there was significant difference in yield among different fertilization treatments from 2016 to 2017, which showed U₂M₂> U₁> M₁> CK. Compared with the U₁ and M₁ treatment in 2016, the yield of U₂M₂ treatment was increased by 3.45% and 5.25%, respectively, and there was no significant difference in yield between U₁ and M₁ treatment. Compared with the U₁ and M₁ treatment in 2017, the yield of U₂M₂ treatment was increased by 5.83% and 12.53%, respectively, and the yield under U₁ treatment was significantly higher than that of M₁ treatment, increased of 6.33%. 【Conclusion】 Compared with single application of urea, organic-inorganic combination application could effectively reduce gaseous loss of nitrogen. Organic-inorganic combination application increased nitrogen use efficiency, dry matter and nitrogen accumulation, and maize grain yield, therefore, it was a reasonable way to increase yield and nitrogen utilization efficiency.

Effects of Pattern of Deep Tillage on Topsoil Features, Yield and Water Use Efficiency in Lime Concretion Black Soil

ZHAO YaLi, LIU WeiLing, CHENG SiXian, ZHOU YaNan, ZHOU JinLong, WANG XiuLing, ZHANG MouBiao, WANG Qun, LI ChaoHai

Scientia Agricultura Sinica. 2018, 51(13):  2489-2503.  doi:10.3864/j.issn.0578-1752.2018.13.005

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【Objective】 The object of the study was to study the effects of subsoiling pattern on topsoil features, crop yield and water use efficiency (WUE), and to identify the suitable tillage system in lime concretion black soil. The results could provide basis for the construction of suitable topsoil of lime concretion black soil. 【Method】 Five treatments, namely, rotary tillage in autumn (ART+SNT, CK), subsoiling in autumn (ASS+SNT), deep mouldboard ploughing in autumn (ADMP+SNT), deep mouldboard ploughing in autumn and sublevel subsoiling in summer (ADMP+SSSS), and deep mouldboard ploughing in autumn and lateral subsoiling in summer (ADMP+SSSL), were carried out to investigate the time and pattern of deep tillage on soil physical property, root growth, crop yield and WUE of winter wheat , summer maize rotation system. 【Result】 Deep tillage significantly reduced soil compaction and soil three-phase R values, promoted roots growth of winter wheat and summer maize, thus increased crop yield and WUE, and the effects of DMP were higher than that of ASS, the effects in winter wheat growth period were bigger than that in summer maize growth period. Compared with ART, the soil penetration resistance of ADMP+SSSS decreased by 20.9%, soil three-phase R value decreased by 12.9%, root dry weight density increased by 29.8%, wheat yield increase by 22.0%, maize yield increased by 8.8%, and total yield of winter wheat and summer maize increased by 15.2%. The WUE during winter wheat growth period, summer maize growth period and whole year increased by 18.2%, 7.9% and 14.0%, respectively, and economic benefits increased by 19.8%. The improvement effects of ADMP+SSS treatment on soil properties were better than that of single deep tillage in autumn. The root growth, yield and WUE of ADMP+SSS were all higher than that of single deep tillage in autumn. Compared with deep tillage in autumn, the soil penetration resistance and soil three-phase R values of ADMP+SSS decreased by 5.6% and 15.0%, respectively. The crop yield and WUE of ADMP+ASS increased by 4.0% and 4.2%, respectively, and economic benefits increased by 3.3%. Among the five treatments, the improvement effect on soil character of ADMP+SSSL treatment was best, and the root growth, crop yield and WUE were highest. The soil penetration resistance and soil three-phase R values of ADMP+SSSL were 28.4% and 26.6%, respectively, higher than that of ART. The root length, root surface area, root volume and root dry weight density of ADMP+SSSL were 67.0%, 45.3%, 23.1% and 49.5%, respectively, higher than that of ART. The yields of winter wheat, summer maize and total yield of ADMP+SSSL were 5.5%, 3.4% and 4.4%, respectively, higher than that of ADMP +SNT; 27.8%, 11.6% and 19.4%, respectively, higher than that of ART. The WUE during winter wheat growth period, summer maize growth period and whole year of ADMP+SSSL were 3.7%, 3.5% and 4.1%, respectively, higher than that of ADMP +SNT; 20.9%, 10.7% and 18.1%, respectively, higher than that of ART. Economic benefit of ADMP+SSSL was 24.4% higher than that of ART.【Conclusion】The crop yield and WUE of ADMP+SSS treatment were higher than that of deep tillage in autumn. Among the five treatments of different deep tillage time and pattern, the crop yield and the WUE of ADMP+SSSL were highest. Thus, the deep mouldboard ploughing in autumn combined with lateral subsoiling in summer was the suitable pattern of deep tillage in lime concretion black soil.

PLANT PROTECTION

Relationship between kynurenine 3-monooxygenase gene BcKMO and cAMP signaling pathway in Botrytis cinerea

YUAN XueMei, WANG Min, ZANG JinPing, CAO HongZhe, ZHANG Kang, ZHANG Jing, XING JiHong, DONG JinGao

Scientia Agricultura Sinica. 2018, 51(13):  2504-2512.  doi:10.3864/j.issn.0578-1752.2018.13.006

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【Objective】The objective of this study is to analyze the relationship between BcKMO and cAMP signaling pathway in Botrytis cinerea, and to lay a foundation for clarifying the molecular mechanism of the BcKMO in growth, development and pathogenicity in B. cinerea. 【Method】 A specific inhibitor SQ22536 of cAMP signaling pathway was used to detect the sensitivity of the wild-type strain BC22, the BcKMO T-DNA insertion mutant BCG183, and the BcKMO complementing mutant BCG183/BcKMO. The cAMP was extracted from the wild-type strain BC22, the BcKMO T-DNA insertion mutant BCG183, and the BcKMO complementing mutant BCG183/BcKMO and detected by using HPLC assay, respectively. Real-time PCR technology was used to detect expression pattern of the BcKMO,the PKA catalytic subunit gene pka1 and pka2, the PKA regulatory subunit gene pkaR, G-protein Gα subunits gene bcg2 and bcg3 in different developmental stages, tissues and culture conditions of BC22. The expression level of cAMP signaling pathway key genes pka1, pka2, pkaR, bcg2, and bcg3 in mutants BCG183 and BCG183/BcKMO was detected by using real-time PCR technology. The expression level of BcKMO in RNAi mutantsof cAMP signaling pathway key genes pka1, pka2, pkaR, bcg2, and bcg3 was detected by using real-time PCR technology.【Result】The BcKMO T-DNA insertion mutant BCG183 was insensitive to the cAMP signaling pathway specific inhibitor SQ22536. The inhibition rate of the cAMP signaling pathway specific inhibitor SQ22536 to mutant BCG183 was significantly lower than the wild-type strain BC22 and the BcKMO complementing mutant BCG183/BcKMO. The cAMP content of the mutant BCG183 was significantly lower than that of the wild-type strain BC22 and the mutant BCG183/BcKMO. The expression pattern of BcKMO, the PKA catalytic subunit gene pka1, G-protein Gα subunits gene bcg2 and bcg3 was basically the same, and the expression level of BcKMO, pka1, bcg2, and bcg3 was higher in 7th day of mycelia and sclerotia of BC22. In addition,the expression level of BcKMO and the cAMP signaling pathway key genes pka1, bcg2, pkaR, bcg2, and bcg3 was higher in BC22 cultured on medium with fructose. The expression level of cAMP signaling pathway key genes pka1, pka2, pkaR, bcg2, bcg3 in mutant BCG183 was significantly higher than that of strains BC22 and BCG183/BcKMO. The BcKMO expression level in the RNAi mutants of pka1and bcg2 was obviously higher than that of BC22, the BcKMO expression level in the RNAi mutants of pka2, pkaR, and bcg2 was obviously lower than that of BC22.【Conclusion】The BcKMO negatively regulated the expression of pka1, pka2, pkaR, bcg2, and bcg3. The pka1 and bcg2 negatively regulated the BcKMO expression, and the pka2, pkaR, and bcg3 positively regulated the BcKMO expression.

The Cause of Influence of Spray Volume on Control Effect of Pesticide Unit Dose in Rice Fields

GU ZhongYan, XU DeJin, XU GuangChun

Scientia Agricultura Sinica. 2018, 51(13):  2513-2523.  doi:10.3864/j.issn.0578-1752.2018.13.007

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【Objective】The objective of this study is to analyze the relationship between the spray volume in rice fields and retention on rice plants, understand the mechanism affecting the control effect of pesticide unit dose, and to provide a basis for the scientific use of pesticides. 【Method】 the liquid retention on unit area of ??rice was measured under spraying conditions, the change of the liquid retention indicated the point of run-off and maximum retention of the liquid on rice leaves. The surface tension of the liquid was measured by the method according to the GB/T 5549-2010, and the critical micelle concentration of the surfactant solution was determined by using the law of the surface tension varying with the concentration of the surfactant. The zisman method was used to measure the critical surface tension of rice leaves. The key factors affecting the leaf capacity of rice leaves were analyzed. By simulating chlorantraniliprole control of rice leaf roller(Cnaphalocrocis medinalis), pymetrozine and chlorpyrifos control of brown planthopper (Nilaparvata lugens) in a spray tower, the relationship between spray volume and droplet density and the effect of pesticide unit dosage on control effect against pests were also investigated. 【Result】 With the increase of the spray volume, the liquid holding capacity of rice leaves increased. After reaching the maximum value, the liquid began to lose and then it decreased to a stable value and no longer changed. The final liquid holding capacity was only 50% of the maximum value. The critical surface tension of the tested rice leaves was 29.90-31.22 mN·m^-1, which meant the tested surface was a low-energy surface. The surface tension of clear water was 71.8 mN·m^-1, which was greater than the critical surface tension of the tested surface. The surface tension of tested rice leaves limited the liquid holding capacity of the leaves. With the additives TX-10 and Silwet-408, the surface tension of the liquid was less than the critical surface tension of rice, which increased the liquid holding capacity of the rice leaves. When the auxiliary agent reached the critical micelle concentration, TX-10 and Silwet-408 provided the best results. The amount of liquid spray affected the density of spray droplets, thus affecting the control effect of pesticide unit dose. When the droplet volume median diameter was 200 μm, the amount of liquid medicine 150 L·hm^-2 was less than 10 drops/cm². The control effect of three effective doses 20, 25 and 30 g of chlorantraniliprole against C. medinalis was less than 60%. The liquid droplet volume 450 L·hm^-2 had less than 40 drops/cm². The control effect of three effective doses against C. medinalis was 56.92%, 62.86% and 65.07%, respectively. The liquid droplet volume 900 L·hm^-2 was 82 drops/cm², the three effective doses of chlorantraniliprole had the best control effect on C. medinalis, which was over 70%. Reducing the droplet volume median diameter and increasing the amount of droplets of unit volume of liquid could reduce the amount of liquid spray. When the diameter of droplets was 75 μm and the liquid volume was 450 L·hm^-2, the droplets were 140 drops/cm² and the control effect of chlorantraniliprole against C. medinalis had no significant difference with 200 μm droplet diameter and 900 L·hm^-2 liquid volume. Because of the barrier of rice canopy, the droplets number under the canopy was very few when spraying on the longitudinal surface of rice. When the droplet volume median diameter was 200 μm and the spray liquid volume was 900 L·hm^-2 or the droplet volume median diameter was 75 μm and the spray liquid volume was 450 L·hm^-2, the amount of droplets under the canopy were less than 20 drops/cm², the pesticides had poor control effect on the N. lugens. Under the canopy spray, pesticides were sprayed directly on the infestation sites of the N. lugens, and the control effect of the pymetrozine and chlorpyrifos unit doses on the N. lugens was significantly improved. 【Conclusion】 The amount of liquid used affects the amount of pesticides deposited on rice plants and the density of droplets per unit area of rice, thus affecting the control effect of pesticide unit doses on pests. When the liquid volume was 450 L·hm^-2 (droplet diameter 75 μm) or 900 L·hm^-2 (droplet diameter 200 μm), the canopy was evenly sprayed up and down in rice fields, which ensured that the liquid retention on rice plant was within the range of the point of run-off and had a sufficient density of droplets, the control effect of pesticide against pests was good.

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

Stability Characteristics of Soil Water-Stable Aggregates Under Different Tillage Treatments in Summer Fallow on the Loess Plateau

LI Hui, DAI XinJun, GAO ZhiQiang

Scientia Agricultura Sinica. 2018, 51(13):  2524-2534.  doi:10.3864/j.issn.0578-1752.2018.13.008

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【Objective】The paper studied the effects of different tillage treatments in summer fallow on the stability characteristics of soil water-stable aggregates in the Loess Plateau.【Method】The 4 rotational tillage experiments, including 4aNT (no-tillage in four experimental years), 3aPT-ST (deep plow in three years, followed subsoiling in forth year), 3aST-PT (subsoiling in three years, followed deep plow in forth year), and 4aST/PT (subsoiling in first year, deep plow in second year, again subsoiling followed by alternating the next year), were conducted in dryland wheat fields of Qiujialing Village in summer fallow period from 2013 to 2017. Based on the experiments, mean weight diameter (MWD), geometric mean diameter (GMD), aggregate stability rate (WSAR), destruction rate (PAD), fractal dimension (D), peak convex coefficient (C_E) and bias coefficient (C_S) were used to analyze and compare soil aggregates stability (＞0.25 mm).【Result】Results indicated that the treatment of 4aPT/ST improved soil organic matter content and winter wheat yield in the dry farmland, and adoption of 4aPT/ST significantly increased the soil aggregates with particles greater than 0.25 mm by 40.4%-45.5%, 61.8%-98.0% and 39.4%-106.1% than 4aNT, 3aPT-ST and 3aST-PT, respectively. In addition, GMD, MWD, WSAR and C_S of water-stable aggregates under 4aST/PT treatment was also higher than that under other three treatments, but D, PAD and C_E of water-stable aggregates under 4aST/PT treatment was lower than that under other treatments at 0-50 cm soil depth (P＜0.05). The correlation between parameters showed that there were significant positive correlation among WSAR, MWD, GMD and C_S, while these indexes had negative correlation with PAD, D and C_E (P＜0.01).【Conclusion】Tillage in summer fallow influenced the stability of soil water-stable aggregates, and adoption of 4aST/PT increased soil macro-aggregate content and stability and improved dry farmland soil structure and yield.

Response of N₂O Emissions to Elevated Atmospheric CO₂ Concentration and Temperature in Rice-wheat Rotation Agroecosystem

WANG Cong, LI ShuQing, LIU ShuWei, ZOU JianWen

Scientia Agricultura Sinica. 2018, 51(13):  2535-2550.  doi:10.3864/j.issn.0578-1752.2018.13.009

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【Objective】This study was conducted to examine the effects of elevated atmospheric carbon dioxide (CO₂) concentration and temperature on nitrous oxide (N₂O) emissions from annual rice-wheat rotation systems, so as to gain an insight into N₂O fluxes response to climate change.【Method】An in-situ field experiment was established in annual rice-winter wheat rotation systems under a T-FACE platform, consisting of four treatments under different CO₂ concentration and temperature levels (ambient CO₂ + ambient temperature, ambient; 500 μmol·mol^-1 CO₂ + ambient temperature, C; ambient CO₂ + temperature increased by 2℃, T; 500 μmol·mol^-1 CO₂ + temperature increased by 2℃, C+T) during 2012-2015. The fluxes of N₂O from rice-wheat rotation fields were measured using static opaque chamber-gas chromatograph method.【Result】(1) On an average of two rice-growing seasons, elevated atmospheric CO₂ concentration significantly increased the biomass and yield of rice by 9.7% and 5.6%, respectively, and those increments of wheat were 11.3% and 5.7% over the three wheat-growing seasons（P＜0.05）, respectively; Elevated temperature significantly reduced the biomass and yield of rice by 21.1% and 31.6%, and those reductions of wheat were 18.0% and 17.7%, respectively; The combination of elevated CO₂ and temperature significantly reduced the biomass and yield of rice by 13.5%（P＜0.05）and 26.0%, and those reductions of wheat were 8.7% and 10.3%（P＜0.05）, respectively. (2) Either elevated CO₂ concentration or temperature did not affect the seasonal patterns of N₂O emission from rice-wheat rotation system. Elevated CO₂ concentration increased N₂O emissions in rice and wheat season by 15.2% and 39.9%, respectively. Elevated temperature did not affect N₂O emissions in rice season, but it significantly increased N₂O emissions in wheat season by 20.5% (P＜0.05). Despite of a considerable interannual variability, N₂O emissions tended to be increased by the combined effect of elevated CO₂ concentration and temperature in rice season; the emissions of N₂O in wheat season were significantly increased by 46.0% under the condition of C+T treatment. (3) The cumulative N₂O emissions in wheat were positively correlated with belowground biomass of wheat and ΔSOC. (4) Elevated atmospheric CO₂ concentration, elevated temperature and their combination increased GHGI of rice-wheat rotation field by 29.1%, 66.3% and 81.8%, respectively.【Conclusion】All of these results showed that both elevated CO₂ concentration and temperature had a strong impact on the emission of N₂O in rice-wheat rotation field. Elevated CO₂ concentration significantly increased the emission of N₂O in both rice and wheat seasons; Elevated temperature significantly increased N₂O emission in wheat season, but no significant change was observed in rice season. Elevated CO₂ concentration increased N₂O-derived GHGI from rice-wheat rotation field, but it was not significantly different; Elevated temperature and the interactive between elevated CO₂ concentration and temperature significantly increased GHGI. The effects of different applied treatments on N₂O-derived GHGI from rice-wheat rotation field from high to low in order were: C+T>T>C. It was suggested from this study that to ensure present crop supply level under the condition of high atmospheric CO₂ concentration and temperature would likely to exacerbate climate change by increasing N₂O emission.

HORTICULTURE

Analysis of the Fingerprints of Different Orange Varieties and Their Differential Metabolites Based on Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry and Progenesis QI

ZHAO XiJuan, ZHAO WuJi, XU HuaChao

Scientia Agricultura Sinica. 2018, 51(13):  2551-2560.  doi:10.3864/j.issn.0578-1752.2018.13.010

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【Objective】The objective of this study is to establish an efficient method to investigate the differences between different varieties of oranges, and to find their differential metabolites, which will serve the citrus metabolomics and provide reference for the identification of oranges and orange juice. 【Method】 8 orange cultivars from the same area were chosen to obtain the methanol extracts of their peels, and then based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS), the LC-MS fingerprints of the 8 oranges were obtained with obvious differences. Combined with the software Progenesis QI, the differential metabolites were screened in positive and negative modes through principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) as well as loadings plots and VIP (variable importance for projection) values. Furthermore, the differential metabolites were identified based on retention times, accurate mass, MS/MS fragments, reference standards and public databases.【Result】Based on the established method, differential metabolites of the 8 orange cultivars were screened. 17 compounds had been identified and among them, and 6 compounds were confirmed by comparison with their commercial standards. 3-Hydroxy-5,7,3',4'-tetramethoxyflavone and luteolin-7-O-neohesperidoside were the characteristic metabolites of Xuecheng. Hesperidin was the marker compound of S26 Jincheng. Narirutin-4'-glucoside and Isosinensetin could act as the characteristic metabolites of 8045 Tiancheng. 【Conclusion】 The method was versatile and suitable for the analysis of sample differences caused by varieties, geographical origins and maturity.

Responses of Leaf Assimilate Export To Lowlight Stress in Melon

YANG LiuYan, CHEN JingJing, CHEN NianLai

Scientia Agricultura Sinica. 2018, 51(13):  2561-2569.  doi:10.3864/j.issn.0578-1752.2018.13.011

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【Objective】The objective of this study is to evaluate the responses of melon leaf photosynthesis, sucrose synthesis and stachyose loading to low light stress during fruit development stage, and the difference in assimilate export between lowlight tolerance and sensitive cultivars, to support the further analysis of sugar unloading and accumulation in fruits of the lowlight tolerant cultivars. 【Method】 A low light-tolerant cultivar (Yujinxiang) and a low light-sensitive cultivar (Yuxuesanhao) of melon (Cucumis melo L.) were used as materials. The melon plants were cultured in greenhouse and were shaded after pollination. And then the mature leaves from the fruit nodes were collected every five days for measurement. The leaf chlorophyll content, gas exchange parameters, levels of soluble sugars (fructose, glucose, sucrose, galactinol, raffinose, stachyose) and starch, activities of sucrose phosphate synthase (SPS), sucrose synthase (SS), acid invertase (AI), neutral invertase (NI), galactinol synthase (GS) and stachyose synthase (STS) were determined. 【Result】 Under shading condition, leaf chlorophyll a/b, net photosynthetic rate (Pn), and contents of glucose, fructose and sucrose in the leaves of both cultivars decreased, but the starch level increased. The reduction of chlorophyll a/b in Yujinxiang leaves (10.0%) was larger than that in Yuxuesanhao leaves (5.8%), but the reduction of leaf Pn, sucrose content and increment of starch content were less in Yujinxiang (30.3%, 30.9% and 3.6%, respectively) than those of Yuxuesanhao (45.2%, 60.6% and 20.4%). Activities of sucrose metabolism enzymes (SPS, SS, AI and NI) decreased after shading, and the reduction of SPS and SS activities were significantly less in Yujinxiang (16.5% and 30.0%) than in Yuxuesanhao (31.6% and 40.5%), but the reduction of AI and NI activities was larger in Yujinxiang (23.8% and 12.7%) than in Yuxuesanhao (18.3% and 1.8%). Galactinol level and GS activity decreased after shading, but no significant genotypic difference was observed. Contents of raffinose and stachyose as well as the activity of STS all decreased after shading. The reduction of raffinose content was larger, the reduction of stachyose content and STS activity was less in Yujinxiang (65.3%, 79.5% and 23.8%, respectively) than those in Yuxuesanhao (35.0%, 83.3% and 32.4%). 【Conclusion】The reduction of leaf sucrose synthesis and stachyose loading after shading was less in Yujinxiang than in Yuxuesanhao, which indicates that the low light tolerant cultivar has stronger leaf assimilate export capacity than the sensitive cultivar under shading. 

FOOD SCIENCE AND ENGINEERING

Analysis of Phenolic Compounds and Antioxidant Activities of Blueberry Leaves from Different Drying Methods

LI XiaoYing, XUE Mei, FAN WenQiao, LUO Jie

Scientia Agricultura Sinica. 2018, 51(13):  2570-2578.  doi:10.3864/j.issn.0578-1752.2018.13.012

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【Objective】This study was aimed to investigate the effects of four different drying processes, including hot air drying (HAD), vacuum heat drying (VHD), microwave drying (MD), and vacuum-freeze drying (VFD), on polyphenols content and antioxidant activities of blueberry leaves, which would provide scientific basis for the comprehensive utilization of blueberry leaves.【Method】The phenolic compounds were determined by Folin-Ciocalteu, NaNO₂-Al(NO₃)_3, and HPLC methods, respectively. Meanwhile, the DPPH radical scavenging capacity, FRAP (ferric reducing antioxidant power), and ABTS⁺· scavenging capacity of the extracts of dry blueberry leaves were determined. The relationship between phenolic compounds and antioxidant activity were also analyzed by correlation analysis. 【Result】The total phenol, chlorogenic acid, and rutin contents were varied significantly in blueberry leaves with different drying methods. The VFD blueberry leaves displayed the highest total phenols (55.7 mg GAE·g^-1 DW) and total flavonoids (104.8 mg RE·g^-1 DW) contents, which were 1.2 times higher than that in MD blueberry leaves, and 3 times higher than that in HAD or VHD blueberry leaves. Additionally, the chlorogenic acid (38.3 mg·g^-1) and rutin (10.2 mg·g^-1) in VFD blueberry leaves were also significantly higher than others, which were 1.5 times higher to MD and 2-6 times higher to HAD or VHD blueberry leaves. Moreover, VFD blueberry leaves showed the strongest antioxidant activity, with the median elimination concentration (EC₅₀) of DPPH and ABTS⁺ radical scavenging capacity at 54.8 µg·mL^-1 and 183.9 µg·mL^-1, respectively, and the FRAP value up to 6.0, the antioxidant activity of VFD blueberry leaves was little bit higher than the MD blueberry leaves (EC₅₀ of DPPH· and ABTS⁺· were 61.8 µg·mL^-1 and 225.7 µg·mL^-1, respectively), while the antioxidant activity of MD blueberry leaves was significantly higher than that of HAD (EC₅₀ of DPPH· and ABTS⁺·were 136.6 µg·mL^-1 and 575.1 µg·mL^-1, respectively, FRAP value 1.7) and VHD blueberry leaves (EC₅₀ of DPPH· and ABTS⁺· were 136.1 µg·mL^-1 and 471.3 µg·mL^-1, respectively, FRAP value 1.8) (P＜0.05). No significant difference were observed on the antioxidant activity between HAD and VHD blueberry leaves. In addition to the rutin, the content of other phenolic compounds was significantly correlated with the antioxidant activity (P＜0.01).【Conclusion】The blueberry leaves processed by VFD method retained the highest content of phenolic compounds, antioxidant activity, and reducing power. However, no significant difference was observed between the VFD and MD methods. Thus, our results suggested that one may choose either VFD or MD method to dry blueberry leaves according to the sample quantity and other specific situations.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Researching on the Effect of the Location of Loxp Sequence on the Gene

XU YingYing, JIN Wei, DAI MinMin, FAN BaoLiang

Scientia Agricultura Sinica. 2018, 51(13):  2579-2591.  doi:10.3864/j.issn.0578-1752.2018.13.013

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【Objective】 In order to get a solid foundation for the site selection of a gene targeting experiment which aimed to realize the co-expression of the foreign gene and the endogenous gene linked through 2A sequence, the effect on the gene expression of different location of the loxp sequence at the open reading frame of a gene need to be clarified when the Cre/loxp system was selected as a tool for reporter gene deletion. 【Method】The enhanced green fluorescent protein (egfp) gene was selected as experimental gene, and red fluorescent protein (dsred2) gene as internal reference gene. Based on pEGFP-N2, pGEM-5zf-loxp plasmid, three plasmids with different location of loxp sequence at the open reading frame of egfp gene named ploxp-EGFP (The loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region of egfp gene), pEGFP-loxp (The loxp sequence located at the downstream of the termination codon in the 3′un-translation region of egfp gene) and ploxp-EGFP-loxp (There is one loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of egfp gene) were constructed. As the internal reference, Plasmid pDsRed2-N1 was co-transfected with constructed plasmids into PK15 cells. Twenty-four hours later, the fluorescence intensity of every transfection was observed and analyzed using fluorescence microscope and software Image J. In order to verify the result obtained using egfp gene as experimental gene, phytase gene was selected as another experimental gene. Based on pIREsNeo, pGEM-5zf-loxp, pT-phytase, pGL4.13[luc2/SV40] plasmid and luciferase gene as expression internal reference gene four plasmids with different location of loxp sequence at the open reading frame of phytase gene named p-SV40-luciferase-CMV-loxp-phytase (The loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region of phytase gene, p-SV40-luciferase- CMV-loxp-phytase-loxp (There is one loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of phytase gene), p-SV40-luciferase-CMV- phytase-loxp (The loxp sequence located at the downstream of the termination codon in the 3′un-translation region of phytase gene), p-SV40-luciferase-CMV-phytase (There is no loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of phytase gene)were constructed. As transfection internal reference, pEGFP-N2 was co-transfection with the differenct plasmid constructed above into PK15 cells, pEGFP-N2 and pDsRed2-N1 were co-transfected into PK15 cells to work as blank control for enzyme activity of phytase and luciferase. Forty-eight hours later, the fluorescence intensity of every transfection was observed and analyzed using fluorescence microscope and software Image J. These data were analyzed using statistics software SPSS19.0. At the same time, the activity of phytase and luciferase of every transfection were measured by Molybdenum Blue Method or using Firefly Luciferase Assay Kit, and these data were analyzed using statistics software SPSS19.0.【Result】In the experiment, the dsred2 gene worked as internal reference gene. Statistics analysis using the average value of red fluorescence of different area of every transfection as basic data shows there is no significant difference between every different transfection. This result shows there is no significant difference in purity of plasmid, handling and cell activity between every transfection. Plasmid electrophoresis result shows there is no significant difference in quality between every plasmid used in transfections, this means the difference in quality between every plasmid used in every transfection is not enough to influence the accuracy for analysis. Analysis on the green fluorescence in the same rule shows there is no significant difference between different transfection of the same plasmid and between pEGFP-N2 and pEGFP-loxp, ploxp-EGFP and ploxp-EGFP-loxp, but there is significant difference between pEGFP-N2, pEGFP-loxp and ploxp-EGFP ploxp-EGFP-loxp. These result shows loxp sequence had no effect on egfp gene expression when it is located downstream of the open reading frame of the gene, but had a negative effect on egfp gene expression when it located upstream of the open reading frame of the gene. Replacing the egfp gene by phytase gene and using egfp gene as transfection internal reference gene, luciferase gene as expression internal reference gene, it shows a same result.【Conclusion】These results indicate that if we try to prepare a transgenic animals in which the foreign gene and the endogenous gene is linked by 2A sequence and co-express under the regulation of the full regulation element of the endogenous gene in one copy manner and the Cre/loxp system is selected as a tool for reporter gene deletion, the downstream of the open reading frame of the endogenous gene is the suitable targeting site.

Evaluation of the Quality of Rectangular Bale Alfalfa Silage

SUN JuanJuan, WANG GuoLiang, A LaMuSi, ZHAO JinMei, BAI ChunSheng

Scientia Agricultura Sinica. 2018, 51(13):  2592-2599.  doi:10.3864/j.issn.0578-1752.2018.13.014

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【Objective】 Rectangular bale silage is one pattern of alfalfa silage, which has a greater flexibility with transport and feeding. The technique of big-bale silage is, however, prone to locally spoilage in the face or in the core, because the plastic bag is easily broken. Evaluation to the bale silage quality would provide a theory basis for choosing the alfalfa silage pattern and its application in feeding. 【Method】 Non-spoilage alfalfa silage samples were taken from rectangular bales which the plastic bag were broken or not broken, from upper layer, middle layer and lower layer. Microbial colony numbers, nutrient composition and fermentation quality were compared. Two evaluation systems of V-Score and Kariser were used to evaluate alfalfa bale silage fermentation quality. 【Result】 The dry matter (DM) content of alfalfa bale silage was 43.86%-45.47%, crude protein (CP) was above 21%, natural detergent fiber (NDF) was 35.87%-37.42%, acid detergent fiber (ADF) was 30.68%-31.79%, Ash was 8.37%-8.50%, and water soluble carbohydrate (WSC) was 0.46%-0.53%. The DM of the sample from alfalfa bale silage which plastic bag were broken (BS) were significantly lower than that of sample from alfalfa bale silage which plastic bag were not broken (NBS). The chemical composition were not different from each other. The pH ranged from 4.36 to 4.72, and LA, AA, PA, BA content was 6.12%-7.04%, 2.41%-3.21%, 0.18%-0.20% and 0.67%-0.89%, respectively. AN/TN was 5.33%-5.79%. pH and AA from BS silage were higher than those from NBS silage. Mold and yeast counts of all sample were lower than 104cfu/FM. The mark of V-Score evaluation was average and the mark of Kaiser evaluation was three. 【Conclusion】 Rectangular bale silage is an good alfalfa silage pattern, and has a good fermentation quality. After removing the visible mold silage, the silage from bale which plastic bags were broken could be used as the silage with same quality of silage from bale which plastic bag were not broken.

Cloning, Expression and Function Analysis of Schistosoma Japonicum ELAV-like 1

XU Rong, ZHANG YuanYuan, LI XiaoChun, CHENG GuiFeng, HE ChuanChuan, GUO Lu, LI Hao, LIU JinMing, GU ShaoPeng, JIN YaMei

Scientia Agricultura Sinica. 2018, 51(13):  2600-2613.  doi:10.3864/j.issn.0578-1752.2018.13.015

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【Objective】 Cloning and expressing of Schistosoma japonicum embryonic lethal abnormal vision like 1(SjELAV-like 1) gene and analyzing the expression status in different stages and at different infected conditions as well as its distributions in the worms of Schistosoma japonicum (S. japonicum). This study also intend to explore the effects of SjELAV-like 1 on the morphology and reproductive development of S. japonicum.【Method】 The RACE technique was used to amplify the 5′/3′ end of the SjELAV-like 1 gene, the obtained sequence was submitted to NCBI, Gene Bank accession number: MG515727. The recombinant plasmid pET28-SjELAV-like 1 was constructed and expressed in E.coli BL21 by IPTG induction, then the products were collected at different time. The SDS-PAGE was used for protein analysis. His-tag nickel column affinity chromatography was used to purify the recombinant protein. The purified recombinant protein was used to immune mouse to obtain the polyclonal antibody, which was used to analyze the immunogenicity of the recombinant protein by western blotting and detect the distributions of SjELAV-like 1 protein in S. japonicum by indirect immunofluorescence assay. The mice infected by cercaria of S. japonicum in the abdomen were used to collect mixture and the females and males separated by paired worms at different stages. The mice infected by cercaria escaped from the single nails were used to collect the unisexual worms at the 25th day. The expression level of SjELAV-like 1 were detected by quantitative real-time PCR (qRT-PCR). The gene silence was performed by tail vein injection of small interference RNA(siRNA) in infected mice. The long-term RNA interference(RNAi) assay was performed to analyze the effects of SjELAV-like 1 gene silence on the spawning and egg hatching of females. The tegument structures and reproductive organs of the females and males of S. japonicum were observed by scanning electron microscopy and transmission electron microscopy after RNAi.【Result】In this study, a 1 797 bp sequence of SjELAV-like 1 was obtained, whose coding region was 1 533 bp encoding 510 amino acids. The expression of the recombinant plasmid reached its highest after 4 h of IPTG induction and it mainly existed in the form of inclusion bodies. High quality polyclonal antibody was obtained against the purified protein. SjELAV-like 1 protein was mainly located in the tegument of the worms. SjELAV-like 1 was expressed in all stages of S. japonicum and the expression was stable in males but gradually became less in females as the maturing of the worms. Expression of SjELAV-like 1 was higher in development impaired females compared with the normal worms. In the RNAi assay, the long-term interference group showed that liver egg burden,liver egg burden by per female and egg hatching rate in interference group were reduced by 58.27% (P＜0.05), 40.59% (P＜0.01) and 74.58% (P＜0.01) compared with NC group, respectively. Electron microscopy observation found that the tegument structures of the worms in interference group were obviously different from that in NC group. The surface bubble adhere to the surface of males disappeared, the three-dimensional fold ridge collapsed, the funicular surface ridge arranged loosely and the network structure of the body wall disappeared. The body surface gap increased and the spines on the body wall of the female were dull. The number of spermatocyte in the males was decreased and the intracellular chromatin was reduced, while the cells were swollen and the intercellular space was widen. In females of S. japonicum the vitelline globules in vitelline cells were decreased, in which the vitelline droplets were also reduced, the endoplasmic reticulum was swollen and the cortical particles were scattered.【Conclusion】Part sequence of SjELAV-like 1 was successfully cloned and expressed, which was robustly expressed in development impaired females. SjELAV-like 1 was mainly expressed in the tegument of S. japonicum. SjELAV-like 1 silence led to decrease of liver egg burden, liver egg burden by per female and egg hatching rate. SjELAV-like 1 silence also altered the tegument structures of the worms and hindered its development of reproductive glands. These all suggested that SjELAV-like 1 play an important role in the development of the reproduction of S. japonicum.

RESEARCH NOTES

Establishment and application of a high-throughout protocol for Peach (Prunus persica) DNA extraction

ZHANG NanNan, NIU Liang, CUI GuoChao, PAN Lei, ZENG WenFang, WANG ZhiQiang, LU ZhenHua

Scientia Agricultura Sinica. 2018, 51(13):  2614-2621.  doi:10.3864/j.issn.0578-1752.2018.13.016

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【Objective】 Preparation of large quantity and high-quality DNA is an important prerequisite for large-scale genotypic screening and molecular marker-assisted of plant breeding. The objective of this study is to present a low-cost, high-throughput peach (Prunus persica L. Batsch) genomic DNA extraction method, meet the needs of high-throughput genetic researches and improve the working efficiency.【Method】The population were obtained from a cross between female parent ‘CN8’ (standard type, ST) and male parent ‘09-1-112’ (temperature-sensitive semi-dwarf in Prunus persica, PpTssd type) to establish a high-throughout protocol for peach DNA extraction. The F₁ segregating population were generated to assess the phenotype characteristics, resulting in observed 1﹕1 (254 standard type and 246 semi-dwarf type individuals). Subsequently, DNA extraction was carried out on the young leaves of two parents and 500 progenies by procedure using 1.2 mL thin-wall 8 strip polypropylene PCR tubes instead of a single centrifuge tube. After extraction, the quality of DNA samples was examined with ultraviolet spectrophotometry and 1% agarose gel electrophoresis, respectively. Referencing the peach genome (version 2.0) and using re-sequencing data, single nucleotide polymorphism (SNP) markers were developed and the HRM analysis was employed on F₁ population to conduct SNP genotyping. Ultimately, the extracted DNA samples were validated by using an InDel marker to verify the genotype of 500 individuals.【Result】The concentrations of DNA were in a range between 25 to 200 ng·μL^-1 and the UV absorbance ratios values (1.81-1.98) to determine DNA quality were acceptable and with high-purity. The result of agarose gel electrophoresis proved that DNA bands were clear, single with a high degree of DNA integrity. Referencing the peach genome and using whole genome re-sequencing data of two parents, SNP_Pp03_3758620 was developed in female and male parents, and the HRM analysis was employed to conduct SNP genotyping and divided temperature-sensitive semi-dwarf and standard type individuals into two groups, respectively, which proved DNA templates extracted from this DNA isolation procedure could be employed for HRM genotyping. Based on the genotype and phenotype of two parents, InDel_Pp03_3829009 was developed and the results of polyacrylamide gel electrophoresis showed that PCR amplification products showed desired fragment size, and were polymorphic in PpTssd type and ST type with bright and clear target fragment. It concluded that the extracted DNA samples could be used for indel analysis. Using this method, 1 000 samples of DNA could be extracted per day with low cost and no effect on the early growth of seedlings.【Conclusion】A simple, effective and low-cost method for extracting genomic DNA from peach was established, which can be used for molecular biology, such as genotyping, variety identification and genetic analysis. Simultaneous extraction of genomic DNA from large quantities of different samples was realized. It has high application value.

Development and Application of SSR New Molecular Marker for Seedless Traits in Grape

MA YaRu, FENG JianCan, LIU ChongHuai, FAN XiuCai, SUN HaiSheng, JIANG JianFu, ZHANG Ying

Scientia Agricultura Sinica. 2018, 51(13):  2622-2630.  doi:10.3864/j.issn.0578-1752.2018.13.017

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【Objective】The objective of this study is to develop a new SSR molecular marker for seedless traits in grape, and to identify the early seedless traits of grape hybrids, and to lay the foundation for molecular marker assisted selection breeding. 【Method】RAD-seq was carried out in 133 grape materials including the female parent ‘Red Globe’, the male parent ‘Centennial Seedless’ and their F₁ hybrids, and the sequencing results were compared to the reference genome. SAMtools software was used to generate CaSFS software required for pileup and GLF files, and the effective SNP sets between parents were obtained by filtration. A window sliding method was used to determine the genotype of each window, and selected one window per 15 SNP and slided one SNP at a time. The genotype of each individual was obtained and the bin generated. Linkage analysis was carried out based on Joinmap software for generated bin, and genetic linkage map was constructed between ‘Red Globe’ and ‘Centennial Seedless’. The QTL loci was mapped using WindowQTLCartographer2.0 software. 35 SSR molecular markers consistent with the SSR characteristic sequence were identified by Perl programming language in the QTL mapping interval, and then 35 pairs of SSR molecular marker primers were designed. The molecular marker was screened in the differences with the seedless traits between parents by HRM technology, and the accuracy of seedless typing, the rate of seedless detection and the rate of seedless holding were detected in 131 F₁ hybrid population and natural population including 65 grape varieties.【Result】On the genetic linkage map between ‘Red Globe’ and ‘Centennial Seedless’, the seedless traits of grape were mapped on chromosome 18, and the mapping interval was 26 835 846-26 960 426, and the contribution rate to seedless was 77.9%, and the LOD threshold was 26.3. The molecular marker VvSD10 was screened in the differences with the seedless traits between parents, and there was a 111 bp loci in seedless traits of grape. Identification of seedless traits in grape F₁ hybrid population and natural population by HRM technology using 111 bp loci on VvSD10 marker, the results indicated that in F1 hybrid population and natural population, the correct typing rate of molecular marker VvSD10 was 97% and 94%, respectively, and the rate of seedless detection of both populations was 56%, the rate of seedless holding was 77% and 85%, respectively. 【Conclusion】Seedless typing in grape by Molecular marker VvSD10 could provide high accurate information in both genetic and natural populations and laid the foundation for molecular marker assisted breeding program in seedless grape.