Scientia Agricultura Sinica

Association Analysis of Leaf Chlorophyll Content with SSR Markers and Exploration of Superior Alleles in Upland Cotton

LIU QiBao, LI LiBei, ZHANG Chi, SU JunJi, WEI HengLing, WANG HanTao, YU ShuXun

Scientia Agricultura Sinica. 2017, 50(18): 3439-3449. doi:10.3864/j.issn.0578-1752.2017.18.001

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【Objective】The objective of this study is to detect the SSR markers associated with leaf chlorophyll content and explore the alleles and their typical materials in upland cotton. The results will be helpful for molecular marker-assisted breeding.【Method】The natural population including 185 upland cotton accessions were planted at two different places in 3 years and the data of leaf chlorophyll content were recorded at 3 stages (0, 10 and 20 days after topping) every year. In analysis the polymorphism information of population structure, Power Marker 3.25 software was used to estimate the polymorphism information content (PIC). The structure of the natural population was analyzed using STRUCTURE 2.3.4 software and the kinship was estimated using TASSEL 3.0 software. Then the data were associated with 137 SSR markers by GLM (general linear model, Q) and MLM (mixed linear model, Q+K). The superior alleles were exploited and the phenotypic effects of total alleles were found.【Result】 Totally 355 polymorphic alleles were found with 137 SSR markers and 2.6 alleles were revealed with each marker in average ranged from 0.01-0.95. The 85% of total alleles were highly polymorphic primers (PIC＞0.5). HAU2146 (PIC=0.95) and NAU2083 (PIC=0.93) kept the maximum PIC. According to the results of STRUCTURE software, K value was 2 when ΔK was the max so the cultivars were divided into 2 populations. A total of 22 alleles found by GLM method significantly at the level of P＜0.001 which explained 5.28%-10.85% of the phenotypic variance and the mean value was 7.24%. SWU0529a (R² =10.85%) and NAU998c (R²=10.48%) kept the max value. Meanwhile, 17 alleles were found by MLM method significantly at the level of P＜0.01 which explained 3.72%-8.58% of the phenotypic variance and the mean value was 4.72%. SWU0923b (R²=8.06%) and SWU0662d (R²=6.74%) kept the max value. A total of 12 alleles were revealed by GLM method and MLM method in common, and NAU998c was significantly at 3 stages by GLM and MLM methods. Two positive alleles (HAU3318b and SWU0987b) were revealed over the estimated phenotypic effects. The 53 carrier materials of two positive alleles kept higher SPAD value in average than the 46 materials carried none of two positive alleles in 10 and 20 days after topping.【Conclusion】A total of 12 alleles associated significantly with leaf chlorophyll of upland cotton were found, and then two positive superior alleles, 53 carrier materials and one typical materials were revealed.

QTL Mapping for Main Root Length and Lateral Root Number in Soybean at the Seedling Stage in Different N, P and K Environments

LIANG HuiZhen, DONG Wei, XU LanJie, YU YongLiang, YANG HongQi, TAN ZhengWei, XU Yang, CHEN XinWei

Scientia Agricultura Sinica. 2017, 50(18): 3450-3460. doi:10.3864/j.issn.0578-1752.2017.18.002

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【Objective】Main root length (MRL) and lateral root number (LRN) are important root traits. It is important to develop the gene resources and reveal the genetic mechanisms of MRL and LRN, and identify quantitative trait loci (QTL) associated with root traits in soybean, including main-effect QTLs, epistatic effects and QTL × environment interactions, meanwhile, map the main-effect QTLs, epistatic effects and QTL × environment interactions in different N, P and K environments. 【Method】A total of 447 RILs derived from a cross between cultivated Jindou23 as the female and native variety HuibuzhiZDD02315 as the male were used as materials. Thirty seeds from each of the RILs and their parents were covered with pasteurized paper, and cultivated in CK (nonfertilized condition), NPK (normal fertilization conditions) and 1.5NPK (high fertilization conditions) at 20-28℃ in 2015 and 2016, and a complete random design with three replications was used in this study. Root traits were measured at V2 stage. Epistatic QTLs and QTL × environment interactions were performed using WinQTLCart 2.5 and QTLNETwork 2.1. 【Result】Twenty-four QTLs for MRL and LRN were detected on chromosomes 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 and 17 using CIM method in this study. The variation accounted for by each of these twenty- four QTLs ranged from 8.52% to 43.62%. These QTLs showed additive effect. Two QTLs for MRL and LRN were detected by MCIM, which showed an additive effect. Another two pairs of additive × additive epistatic effects QTLs for MRL and LRN were detected, including one pair of major QTLs and non-major QTL additive × additive epistatic effects, and one pair of non-major QTLs and non-major QTL additive × additive epistatic effects. Two pairs of QTL interaction for MRL explained 1.53% and 1.95% of the phenotypic variation, and two pairs of QTL interaction for LRN explained 2.47% and 1.13% of the phenotypic variation. Two QTLs were simultaneously detected on the same chromosome using two WinQTLCart 2.5 and QTLNETwork 2.1. Nine QTLs were simultaneously detected in three environments. The QTL for MRL was all mapped on chromosome 6 in 2015 (including NPK and 1.5NPK) and 2016 (including 1.5NPK). The QTL for LRN was all detected on chromosome 5 in 2015 (including NPK and 1.5NPK) and 2016 (including CK), another QTL for LRN was all mapped on chromosome 17 in 2015 (including CK and NPK) and 2016 (including NPK). 【Conclusion】MRL and LRN absorb less NPK at seedling stage in soybean, so farmers should minimize the use of NPK in agricultural production. MRL and LRN were controlled by the same controlled gene and specific gene in NPK treatments. Some QTLs were not simultaneously identified in different NPK environments as the related genetic mechanism is comparatively complex. Additive effects, additive × environment interactions and additive × additive epistatic effects are important genetic factors in MRL and LRN formation and inheritance. One each QTL for MRL and LRN was all detected by CIM and MCIM; one stable gene for MRL and LRN existed in interval markers between Satt442-Satt296 and Satt521-GMABABR.

Evaluation of Low-Light Tolerance of Different Potato Varieties and Screening of the Evaluation Indexes

LI CaiBin, GUO HuaChun

Scientia Agricultura Sinica. 2017, 50(18): 3461-3472. doi:10.3864/j.issn.0578-1752.2017.18.003

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【Objective】 In recent years, the low light stress has become an important limiting factor for the development of winter-cropping, protected cultivation and intercropping potato production. The objective of this study is to screen the identification indices of potato low light tolerance, and to evaluate the low light tolerance of different potato varieties, so as to provide a scientific basis for the low light tolerance potato breeding, winter-cropping and intercropping potato production. 【Method】 In a field experiment, 10 potato cultivars were used as the test materials and planted. The plants were shaded with black nets which had 70% shading rate of natural light from emergence to harvest. The natural light plants were used as the control. The plant heights, internode elongations, leaf areas and other morphological indexes, pigment contents including chlorophyll a, chlorophyll b and carotenoid and net photosynthetic rates, stomatal conductance, transpiration rates and other photosynthetic physiological indexes were measured at bud flowering stage. Yields were tested after harvest. In order to make it can be comparable among these indexes, the low-light tolerance coefficient, low-light tolerance index and each shade stress index were calculated by varies of yields and other traits, then the low-light tolerance coefficients and low-light tolerance indexes expansion values were used to evaluate low-light tolerance types of different potato varieties. After that, correlation analysis were performed according to the indexes of stress index respectively with low-light tolerance coefficients and low-light tolerance indexes, screening significant related indicators. 【Result】 The potato varieties were changed in morphology, physiology, and yield after shading treatment. Compared with the control, plant heights, internode elongations and the leaf areas were increased; the chlorophyll a, chlorophyll b and total chlorophyll contents were increased, the chlorophyll a/b values were decreased, and the carotenoid contents showed an increasing trend; the net photosynthetic rates, instantaneous water use efficiencies, and stomatal conductance were decreased, the transpiration rates were increased and intercellular CO2 concentrations showed an increased trend; tuber weights were decreased significantly by 62.14%-90.74%. The assessment results showed a similar trend in tolerance by low-light tolerance coefficients method and low-light tolerance index method. Low-light tolerance index not only reflects the potato yield sensitivity to shading stress, but also reflects the influences of different genotype differences on the yield, the evaluation results of the low-light tolerance index was more strict than the method by low-light tolerance coefficient. There are 6 stress indexes including leaf area, per plant tuber weight, chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content were significantly or extremely significantly correlated with the low-light tolerance coefficient or low-light tolerance index, which can be used as the effective indicators to identify the low-light tolerance of potato.【Conclusion】 After comprehensive evaluation, it was concluded that the ‘Zhongshu20’ has a high low light tolerance, ‘Woff’ has a moderate low light tolerance, ‘Favorita’ and ‘Hui-2’ have a poor low light tolerance, and other potato varieties have a bad low light tolerance. The way to use the low-light tolerance coefficient and low-light tolerance index as the main index, and combined with the related morphological, physiological and yield indexes for evaluation of the low-light tolerance of potato varieties is a more objective, reliable and convenient method. Key words:

Correlation Analysis on Rutin Accumulation and Gene Expression of Rutin Synthetic Enzymes and MYBs in the Whole Developmental Stage of Fagopyrum tataricum

SUN ZhaoXia, HOU SiYu, LINGHU Bin, LIU RongHua, WANG Li, YANG WuDe, HAN YuanHuai

Scientia Agricultura Sinica. 2017, 50(18): 3473-3481. doi:10.3864/j.issn.0578-1752.2017.18.004

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【Objective】 To explore the relationship among the rutin content at different developmental stages with rutin biosynthesis genes and MYB regulation genes, it will be very meaningful to understand the underlying molecular metabolism of rutin accumulation in tartary buckwheat. 【Method】 The whole developmental stages of F. tatarium Jiujiang were classified into 8 stages (germination, first pair of leaf formation, true leaf growth, vegetative growth, flowering, peak flowering, immature seeds , matured seeds),which were named S1-S8 stages. The whole seedlings from S1and S2 stage, leaves from S3 and S4 stage, flowers from S5 and S6 stage, seed formation organ from S7 and S8 stage were used as test materials. Two MYB genes, FtMYB7 and FtMYB9, were cloned and sequenced. The homologous amino acid sequence and conservative structure domain analysis were carried out by T-coffee software. The phylogenetic tree was constructed by the NJ method (neighbor-joining). The multiple sequence alignment of MYB transcription factors related with flavonoids among Arabidopsis and buckwheat were analyzed by MEGA 7.0 software. The rutin content of these tissues at S1-S8 stages was detected by HPLC. In the meantime, the expression level of rutin-biosynthesis-related genes (FtCHS, FtF3H, Ft4CL, FtFLS-like and FtUFGT) and regulator genes (FtMYB7 and FtMYB9) were investigated by qRT-PCR. The correlation of rutin content and the gene expression pattern during the whole developmental stage was estimated by Pearson correlation method. These data were transformed to matrix data, and constructed a Hierarchical cluster heat map by Euclidean distance method. 【Result】Cloned two MYB transcriptional factors, FtMYB7 and FtMYB9, their nucleic acid sequences were 876 and 912 bp, respectively, encoding 291 and 303 amino acid residues. The sequences of 17 MYB transcription factor from buckwheat, and 3 genes related to flavonoids metabolism in Arabidopsis were constructed for a phylogenetic tree. The phylogenetic tree showed that these genes were divided into six groups. Among them, FtMYB7 belongs to the group II, and FtMYB9 belongs to IV groups, the result indicated that the two MYB transcription factors involved in function diversity at regulation of plant growth and development process. The qRT-PCR results showed that the relative expression levelsof FtMYB7 at S3 and S5 stages were the highest at 501 and 867 times, respectively. To FtMYB9, the highest gene expression levels were detected at S1 and S2 stages, and 34 and 72 times, respectively. Correlation analysis showed that during 8 growth stages, Ft4CL, FtCHS, FtF3H, FtUFGT and FtMYB7 expression patterns were positively correlated with rutin content, and the correlation coefficient was 0.748, 0.683, 0.704, 0.890 and 0.862, respectively. However, FtFLS-like and FtMYB9 were negatively correlated with rutin content, and the correlation coefficients were -0.442 and -0.501, respectively. 【Conclusion】 The FtMYB7 and FtMYB9 were characterized by two R2R3 MYB genes. The different gene expression patterns and rutin content at the whole developmental stage of tartary buckwheat suggested that the FtMYB7 is positively regulated in rutin biosynthesis, but the FtMYB9 is negatively regulated.

Effect of Vertically Cutting Roots at Different Horizontal Distances from Plant on Leaf Photosynthetic Characteristics and Yield of Summer Maize with Different Root Types

LU DuXu, XU ZhenHe, LIU Mei, LIU Peng, DONG ShuTing, ZHANG JiWang, ZHAO Bin, LI Geng, LIU ShaoKun, LI QinFang

Scientia Agricultura Sinica. 2017, 50(18): 3482-3493. doi:10.3864/j.issn.0578-1752.2017.18.005

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【Objective】This study was conducted to evaluate the regulating effects of root in different distances to plant center on leaf photosynthetic characteristics and yield, aimed to provide theoretical supports for screening ideal root system architecture to obtain further yield increase. 【Method】Two summer maize cultivars with different root types, Denghai 661 (DH661, deep root type) and Zhengdan 958 (ZD958, shallow root type) were chosen as experimental materials. At the V12 stage, roots were cut vertically at different horizontal distance of 10 cm (T-10), 20 cm (T-20) from maize plant center at 60 cm soil depth, while no roots cutting as control (CK), to investigate the role of root in different soil profiles in regulating yield and leaf photosynthetic characteristics. 【Result】Results shows that, roots of these maize varieties were mainly located within a horizontal area 0-10 cm from the plant center and in this area, 83.13% of total root biomass, 69.55% of total root length, 68.74% of root surface area and 66.44% of root volume were located for DH661, but for the ZD958, percentage of these index were 75.19%, 51.17%, 53.85%, 56.49%, respectively. Compared to ZD958, the root distribution in horizontal of DH 661 was more compact and less impact caused by cutting root in both sides of plant in the distance of 10 cm or 20 cm to plant center. Roots excision made a prominent impact on the photosynthetic characteristics of the two maize varieties leaves, being followed by inhibiting the growth of leaves before anthesis and accelerating senescence after bloom. Root pruning reduced chlorophyll content of two maize varieties and net photosynthetic rate in two years, at tasseling stage (VT), the chlorophyll content of T-20 treatment of DH661 decreased by 4.29% and 6.32%, respectively, the net photosynthetic rate decreased by 5.16% and 4.66%, the reduced rate of T-10 were 6.37% and 6.86%, 6.47% and 8.66%. The reduced photosynthetic rate of T-20 treatment of ZD958 were 6.52% and 9.91%, 6.48% and 9.15%, in T-10 treatments were 15.40% and 15.01%, 11.89% and 15.49%. At the same time, resulted in significant root shoot biomass and grain yield decreased, the index decline was T-10 > T-20, and the deep root type of DH661 was significantly less than the shallow one ZD958. 【Conclusion】Cutting the roots vertically decreased the ability of net photosynthetic of ear leaf, shoot biomass and final yield of the two maize varieties with different root architecture significantly. The range of reduce increases when the cutting position is close to the main stem. The impact of T-20 on DH661 is obviously weaker than that on ZD958. Compare the root architecture, distribution and the root ability of two maize reserved after cutting vertically, the root performance of deep type is more adaptable to greater planting density.

Morphological and Physiological Mechanism of Salt Tolerance in Gossypium barbadense to Salt Stress at Seedling Stage

ZHAO HaiYan, WANG JianShe, LIU LinQiang, CHEN YongQuan, LI MengFei, LU QuanWei, LIU Fang, PENG RenHai

Scientia Agricultura Sinica. 2017, 50(18): 3494-3505. doi:10.3864/j.issn.0578-1752.2017.18.006

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【Objective】The changes of cell structures and physiological characteristics in leaves and stems of two Gossypium barbadense varieties, Yuehai 9 (salt tolerance) and PS-7 (salt sensitive), were investigated under different salt stress conditions. Morphological and physiological mechanism of salt tolerance in Gossypium barbadense to salt stress at seedling stage was studied to offer a theoretical basis and technical reference for selecting salt-tolerant genes in cotton cultivars. 【Method】In a water culture experiment, three-leaf cotton seedlings were treated with NaCl at 300 mmol·L^-1 NaCl for different times. This study involved observation of morphological features and comparison of physiological indexes of two Gossypium barbadense, Yuehai 9 (salt tolerance) and PS-7 (salt sensitive) under different salt stress treatments by means of optical microscopy and measurement of physiological indices.【Result】There was a difference in effect on two cotton cultivars by salt stress. At 300 mmol·L^-1 of NaCl, with the prolongation of treatment period, the transections of the leaves and stems of Yuehai 9 and PS-7 were both significantly smaller compared with the control groups, decreased, respectively, by 14.10%, 54.69% and 45.30%, 87.90% at 300 mmol·L^-1 of NaCl after 24 h, and the variation range of PS-7 was larger than that of Yuehai 9. The xylem of the vascular bundle of PS-7 was severely damaged while the xylem of the vascular bundle of Yuehai 9 showed no significant change in 300 mmol·L^-1 of NaCl treatment for 12 h. The long cylindrical palisade tissue cell of PS-7 became smaller and ovoid in 300 mmol·L^-1 of NaCl treatment after 24 h while the shape of the palisade tissue cell of Yuehai 9 showed no change. The chlorophyll contents, superoxide dismutase and peroxidase activities in leaves of Yuehai 9 were significantly higher than those of PS-7, while its malondialdehyde contents was lower than that of PS-7. Yuehai 9 and PS-7 were different in chlorophyll contents, malondialdehyde contents, superoxide dismutase and peroxidase activities, and the differences reached a significant level at 300 mmol·L^-1 of NaCl after 8 h. 【Conclusion】 The most sensitive plant part to salt stress may be the palisade tissues of leaves and xylem in the stem. The chlorophyll, malondialdehyde, superoxide dismutase and peroxidase can be used as the physiological indexes for evaluating and selecting salt tolerance cotton materials.

Spatial-Temporal Characteristics of Cropland in the Ugan-Kuqa River Delta Oasis

Maieryemu Yasen, Maimaiti Shawuti, Tuerxun Aishan, Ruzemaimaiti Mijiti, Yikeliman Abudumiti, Mayila Reheman

Scientia Agricultura Sinica. 2017, 50(18): 3506-3518. doi:10.3864/j.issn.0578-1752.2017.18.007

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【Objective】Spatial and temporal characteristics of cropland are a main part of the LUCC research and it is becoming more important for food security, land protection and environmental change. XinJiang plays a significant role for the protection and sustainable development of the land resource which is ranked the richest province in China, with the agricultural land. The Ugan-Kuqa river delta oasis is considered the most important “food and cotton resource area” which is the largest irrigated region in the Aksu district in XinJiang. The main objective of this study is to analyze the causes of spatial and temporal change of the cropland in the research area, from 1994 to 2015 over 21years period. 【Method】 The cropland data were obtained from the Landsat images after processing based on the remote sensing platform. The changing rate, location index and plate density were selected for the analysis of the spatial and temporal changing dynamics of the cropland in recent 21 years for the research area. 【Result】 (1)The results showed that significant changes in the distribution and extent of cropland were occurred that it covered a surface area of 2 488 km² (17% of the total area) in 1994, 3 931 km² (26%) in 2015 with an annual increasing rate of 2.7% (68.71 km²·a^-1).The increased cropland mostly distributed in the margins of oasis and expanded to desert regions from the middle and upper reaches of the oasis. It could be attributed to agricultural activities and urban construction for increasing cropland and residential land, while the saline land and water land largely decreased; (2) Location index presented that there was a significant regional variation in 21 years period. The rank of location index was in the following order: Xinhe＞Shaya＞Kuqa, location index of the Xinhe and Shaya’ counties was larger than mean value of the total area, becoming more concentration in distribution.【Conclusion】Cultivated landscape is becoming continuous from messy pieces over a 21-year period. These changes are due to the increased precipitation and runoff with supplying more water resource. Hence, population growth and its pressure on the land cultivation, improved drainage system are the main reasons for the growing of cropland. The methodology put forward in this study enables us to apply easily and quickly remote sensing and GIS techniques for detecting spatial and temporal change of cropland.

Screening of the Host Factors of Woodland Strawberry Interacting with P6 of Strawberry vein banding virus by Yeast Two-Hybrid System

LI Shuai, JIANG XiZi, LIANG WeiFang, CHEN SiHan, ZHANG XiangXiang, ZUO DengPan, HU YaHui, JIANG Tong

Scientia Agricultura Sinica. 2017, 50(18): 3519-3528. doi:10.3864/j.issn.0578-1752.2017.18.008

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【Objective】 Strawberry vein banding virus (SVBV) is a main virus infecting woodland strawberry (Fragaria vesca), but the SVBV infection mechanisms on woodland strawberry remains unknown. The objective of this study is to provide a theoretical basis for studying the SVBV infection mechanisms on woodland strawberry, SVBV P6 was used as a bait protein to screen the host factors from the cDNA library of woodland strawberry. 【Method】 Woodland strawberries were inoculated with SVBV, and total RNA was extracted from the leaves showed obvious disease symptoms. The total RNA was treated with DnaseI and double-stranded cDNA was synthesized using SMART technology. cDNA was treated with homogenization and enzymatic digestion, and the short fragments with length less than 400 bp were removed. Then the other cDNA fragments were ligated to plasmid vector pGAD-T7 to construct the primary cDNA library of woodland strawberry. Simultaneously, SVBV P6 was ligated into the yeast two-hybrid bait vector pGBK-T7, and the plasmids of pGBK-P6 and pGBK-T7 were transformed into AH109, respectively. The positive yeast clones were grown in the SD/-Trp liquid medium for identifying the toxicity of pGBK-P6 on the yeast AH109. The yeast transformed with pGBK-P6 was grown on the plate of SD/-Trp, SD/-Leu-Trp and SD/-His-Trp medium, respectively, and then the growth situation of the yeast was tested and the self-activating effect of pGBK-P6 on the reporter gene of yeast was analyzed. Then the AH109 containing bait vector pGBK-P6 was transformed with the primary cDNA library of woodland strawberry, the co-transformed yeasts were coated on the plate of SD/-Leu-Trp, SD/-Leu-Trp-His and SD/-Trp-Leu-His-Ade/X-α-Gal medium in turn. Finally, the blue and well grown positive clones were selected. The plasmids of positive yeast clones were extracted and sequenced. The candidate genes were preliminarily compared in the GenBank, and the interacted protein factors were annotated and the protein’s biological functions were analyzed with gene ontology (GO) pathway of Uniprot online websites. 【Result】 Three libraries with the average capacity more than 2.0×10⁶ cfu were constructed, and the average library recombination rate was 97% and the average amplification sizes of inserts fragment of cDNA library were above 1 kb. It demonstrated that the cDNA library of woodland strawberry measured up to the experiment standard. The 230 positive clones were finally selected by using the SD/-Trp-Leu-His-Ade/X-α-Gal medium. After sequence similarity comparison, removing the repetitive sequences, the vector sequences and the frameshift sequences, the 15 host factors interacted with SVBV P6 were screened. GO pathway annotation showed that the 15 host factors were involved in 13 biological processes, including protein ubiquitination, regulation of transcription factor process, defense response, protein catabolic process, oxidation-reduction process and cellular amino acid metabolic process, etc. Moreover, molecular functions of the 15 host factors are mutiple, including acetyltransferase activity, terpene synthase activity, dehydrogenase activity, metalion binding activity, protease activity and hydrolase activity, etc.【Conclusion】The cDNA library of woodland strawberry was constructed successfully, and 15 host factors of woodland strawberry interacted with SVBV P6 were preliminarily screened. This work can provide a theoretical basis for further exploring the molecular interaction mechanism between SVBV and woodland strawberry.

Identification of β-COP and Sro of Tetranychus cinnabarinus and Their Lethal Effects After Silencing

AO YiQianYun, SHEN GuangMao, WANG MengYao, LIU JiaLu, PAN Yu, HE Lin

Scientia Agricultura Sinica. 2017, 50(18): 3529-3539. doi:10.3864/j.issn.0578-1752.2017.18.009

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【Objective】 The objective of this study is to clarify the molecular information of Sro and β-COP genes of Tetranychus cinnabarinus, using the optimized RNAi system to study the lethal effects of these two genes on the T. cinnabarinus, and to provide a basic information to identify target genes, which are useful in the application of RNAi control. 【Method】 Firstly, full sequences of the two genes were identified and the bioinformatics were analyzed by homologous alignment of the sequence, prediction of the conserved region, protein structure and construction of the phylogenetic tree. Secondly, the RNAi system was improved by reducing the factors that will degrade the dsRNA, timely replenishment of dsRNA and other methods, and the gene silencing effect of the new RNAi system was evaluated by quantitative PCR technique. Finally, based on the optimized RNAi system, the expressions of β-COP and Sro genes were silenced to evaluate the lethal effects, and the lethal phenotypes were observed. 【Result】The length of open reading frame of β-COP is 2 688 bp, encoding a 895 aa protein, which belonging to “WD40 superfamily” and “Coatomer_WDAD superfamily”, containing the conserved regions of “WD 40” and “Coatomar_WDAD”. The length of open reading frame of Sro is 1 119 bp, encoding a 372 aa protein. It has two characteristics of typical short chain dehydrogenase: one is binding sequence of NADP+biding binding sequence “TGxxxGx”, the other is “YxxxK” Motif and its upstream aspartic acid (Asn), serine (Ser) active sites. The optimized RNAi could maintain the silencing efficiency of about 50% after 96 h. Based on this system, the expressions of β-COP and Sro were silenced and the death rate showed significant differences between experimental group and control group after 48 h. β-COP dsRNA caused 57.4% mortality rates at 108 h post-treatment, and Sro dsRNA caused 28.8% death rates at 96 h post-treatment. In addition, the phenotype of dead individuals in the β-COP group showed that the four pairs of feet were curled up on the body side and died, and in the Sro group, the mites dead during the molting process. 【Conclusion】The optimized RNAi technique has stable and continuous silencing effect on the T. cinnabarinus. The combination of β-COP and Sro genes functional verification with RNAi technique showed that the silencing of the two target genes can produce a certain lethal effect on the mite. It also shows that the combination of gene and technology has great potential for development and utilization, which can lay a basis for the future functional verification, screening gene and developing the RNAi technology to control the T. cinnabarinus.

Effects of Long-Term Application of Chemical and Organic Fertilizers on Root Biomass and Nutrient in Double Cropping Rice System

LIU KaiLou, ZHANG HuiMin, HAN TianFu, ZHOU LiJun, LI DaMing, HU ZhiHua, HUANG QingHai, YE HuiCai, XU XiaoLin, HU HuiWen

Scientia Agricultura Sinica. 2017, 50(18): 3540-3547. doi:10.3864/j.issn.0578-1752.2017.18.010

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【Objective】 Rice root returned to soil not only promotes mineral nutrients returned to soil, but also improves soil fertility. However, little information is reported about the root biomass and root nutrients accumulation. 【Method】 Based on the long-term field experiment started from 1981, four treatments were chosen, (1) no fertilizer treatment (CK), (2) nitrogen (N), phosphorus (P) and potassium (K) fertilizers (NPK), (3) Double rates of NPK fertilizers (2NPK), (4) NPK + manure (NPKOM). The soil and plant were sampled and measured in 2012. The changes of rice root biomass and their N, P, K contents were analyzed, then, the response of soil fertility factor to root were explored. 【Result】 Rice root biomass, nutrient accumulation and their proportions in whole plant were shown that early rice was significantly higher than that of late rice among all treatments, meanwhile, application of chemical and organic fertilizers increased significantly root biomass and nutrient accumulation in both rice growing seasons than other treatments. Compared with NPK, the root biomass and N, P and K accumulation in early rice growing season increased by 20.58%, 17.34%, 270.66% and 37.89%, respectively; and correspondingly the values in later rice season were 23.85%, 17.88%, 262.55% and 37.96%, respectively. However, compared with early rice, the proportions of root biomass, N and K accumulation in whole plant in NPKOM treatment in the late rice growing season decreased by 22.18%, 33.90% and 26.70%, respectively; and correspondingly the values in NPK treatment were 4.91%, 39.41% and 27.06%, respectively. Conversely, the proportions of root P accumulation in whole plant in NPKOM treatment in late rice growing season showed an increase tread in comparing with early rice growing season. Further, its multivariate regression analysis showed that soil total P and available P contents were key factors in the increase of rice root biomass and the accumulation of N, P, K in root. 【Conclusion】 Long-term application of organic and inorganic fertilizers increased rice root biomass and nutrient accumulation, but decreased the proportions of rice root N and K accumulation in whole plant. Moreover, soil P content played an important role in rice root growth and nutrient accumulation.

Grain Yield and WUE Responses to Different Soil Water Storage Before Sowing and Water Supplies During Growing Period for Winter Wheat in the Loess Tableland

LI Chao, LIU WenZhao, LIN Wen, HAN XiaoYang, ZHOU Ling, WANG YaPing

Scientia Agricultura Sinica. 2017, 50(18): 3549-3560. doi:10.3864/j.issn.0578-1752.2017.18.011

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【Objective】A field experiment was carried out to study the influence of soil water storage before sowing and different water supplies (precipitation + supplemental irrigation) during a growing period on wheat yield, water consumption and water use efficiency, to reveal the regulation mechanism of effective utilization of limited water resources in farmland, and to make clear the achievable level of dryland production potential of winter wheat under the existing measures in the Loess Tableland.【Method】Field experimental plots were designed as follows, based on different soil water contents achieved by either covering the plot or growing a crop in the summer fallow period. (1) In order to analyze the response of wheat yield and water use efficiency to the change of soil water storage before sowing, four rain-fed treatments with different soil water storage levels were formed in the whole growth period of winter wheat. The water storage in 2 m soil profile changed in the range of 350 - 550 mm.（2）Under the same average soil water storage of 500 mm, 3 respective treatments with one time irrigation of 40 mm at jointing stage, booting stage or filling stage were applied to explore different responses of equal irrigation at different growth stages of winter wheat.（3）Under the high water storage levels of 542.3 mm and 571.6 mm, treatments of 2 times and 4 times irrigation were carried out, respectively, to study the possible increase of winter wheat yield and the features of water use efficiency under low water stress.【Result】(1) Under the seasonal distribution of precipitation characteristics of Loess Plateau, the soil water storage before sowing had a decisive effect on wheat yield, and wheat yield increases linearly with the soil water storage. When soil water is well conserved in the summer fallow period, the yield of winter wheat in dryland farming can reach 88% - 90% level of wheat yields under sufficient water supply.（2）Compared with the treatment of 500 mm water storage in the 2 m soil profile and no supplemental irrigation, per increase of 40 mm water supply yield increased by 11.8% for the soil water storage increase, followed by irrigation at the jointing stage and booting stage respectively. However, no significant differences between the three treatments were detected. The winter wheat yield of the treatment with higher soil water storage before sowing and 2 times irrigation in both the jointing stage and booting stage reached a high yield level in the test years with a high WUE. (3) There was a Logistic relationship between yield and water consumption. With the increase of water consumption, the yield increased fast at first and then slowly; the marginal water use efficiency (MWUE) declined continuously; WUE showed three stages of change: rising, peak and falling; and the water consumption at the maximum WUE was less than that at the maximum yield. 【Conclusion】Under the climate condition of the Loess Plateau, both the soil water storage before sowing and different water supply during a growing period influenced the yield of winter wheat. Equal incremental water supply by soil water storage or irrigation in different growth periods had basically the same effect on yield when the total water supply was the same. The relationship between yield and water consumption of winter wheat could be simulated by a Logistic curve model that can be used to reveal the intrinsic relationships among yield, water consumption and WUE.

Effects of Different Controlled Irrigation Low Limits on the Size Distribution of Soil Aggregates with Drip Irrigation Under Film Mulching in a Greenhouse Soil

MA JianHui, YE XuHong, HAN Bing, LI Wen, YU Na, FAN QingFeng, ZHANG YuLing, ZOU HongTao, ZHANG YuLong

Scientia Agricultura Sinica. 2017, 50(18): 3561-3571. doi:10.3864/j.issn.0578-1752.2017.18.012

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【Objective】 Irrigation is the main source of soil moisture and affects the soil structure stability under greenhouse condition. The objective of this paper is to explore the effects of different controlled irrigation low limits on composition and stability of soil aggregates in a greenhouse soil. Results of the study will provide a theoretical basis for reasonable water regulation and improvement soil structure.【Method】The irrigation experiments were conducted in a greenhouse at the scientific research base of Shenyang Agricultural University, China during the period from 2011 to 2016. The test crop was tomato, and the planting mode was ridge and furrow covering. Different controlled irrigation low limits were set to: 20 kPa (D20), 30 kPa (D30), 40 kPa (D40), respectively, and the irrigation allowable upper limit of them was set to 6 kPa. Soil water suctions detected by tensiometers were placed in 30 cm soil layer. The aggregates amount, macro-aggregate content (R_0.25), geometric mean diameter (GMD), mean weight diameter (MWD), structure deterioration rate (RDS), unstable aggregates index (E_lT)and fractal dimension (D) were examined by dry and wet sieving methods. 【Result】 In the 0-30 cm soil layer, the electrical conductivity (EC), cation exchange capacity (CEC) and soil bulk density in D40 treatment were significantly lower than those of D20 and D30 treatments (D40＜D30＜D20). The pH value of D20 treatment was significantly lower than that of D30 and D40 treatments (P＜0.05). In the 0-30 cm soil layer, the size class of dry-stable aggregate was mainly composed of ＞2 mm and 1-0.25 mm (23.01%-39.98%); the size class of water-stable aggregate was mainly composed of 1-0.25 and 0.25-0.053 mm (31.08%-47.27%). In the 0-20 cm soil layer, R_0.25, GMD and MWD in D30 treatment were significantly higher than those of D20 and D30 treatments. While in the 20-30 cm soil layer, the wet stable aggregate content of D20 treatment was significantly higher than that of D30 and D40 treatments (P＜0.05). The soil structure deterioration rate (RDS) and unstable aggregate index (E_lT) increased with soil depth under different irrigation control limits, and the variation of RDS and E_lT was similar. In the 0-20 cm soil layer, the RDS and E_lTof D30 treatment were significantly lower than those of D20 and D30 treatments. But in the 20-30 cm soil layer, the RDS of D20 treatment was lower than those of D20 and D30 treatments by 12.2% and 16.8%, respectively. By dry sieving, the soil fractal dimension (D) of D20, D30 and D40 in the 20-30 cm soil layer was 2.13, 2.08 and 2.19, respectively; by wet sieving, that of D20, D30 and D40 treatment in the 20-30 cm soil layer was 2.31, 1.99 and 2.12, respectively. Overall, compared with D20 and D40, D30 treatment significantly reduced the fractal dimension (D) in the aggregates.【Conclusion】 The result suggests that the 30 kPa drip irrigation treatment greatly enhanced the composition of macroaggregates and the stabilization of soil structure.

Fine Mapping of Dwarfing Gene for Peach Based on SNP Markers

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

Scientia Agricultura Sinica. 2017, 50(18): 3572-3580. doi:10.3864/j.issn.0578-1752.2017.18.013

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【Objective】 Dwarfing peach, due to the small trees, shorter internodes, is an important genetic resources in ornamental and rootstock breeding program. The fine mapping of peach dwarfing genes and explication of the genetic mechanism will provide a basis for the establishment of a molecular-assisted selection system and desired variety improvement, which could be used in breeding dwarf ornamental peach and rootstock varieties. 【Method】 Based on the results of genetic analysis in this study, 395 individuals generated from a self-pollinated population of 05-2-144 were selected for fine mapping the dwarfing gene in peach (Prunus persica (L.) Batsch). By referencing the peach genome sequence, SNP markers were developed in the parents and progenies to generate markers linked to the locus and used to map the gene based on Sanger sequencing. Subsequently, the parent was resequenced to generate SNP markers in the mapping region and acquire heterozyous SNPs for fine mapping. Within the fine mapping region, SNP primers were designed to verify the phenotype of 89 individuals generated from the F₁ segregation population of 10-7×96-5-1.【Result】The segregating population of 05-2-144 was generated to assess the genetic characteristics, resulting in observed 3:1 (300 standard type and 95 dwarfing type individuals) Mendelian ratio fitting with the expected ratio for a monogenic recessive genetic control (P=0.67, χ²=0.19). Moreover, the ratio of standard type to dwarf type corresponded to the expected 3:1 (66 standard type and 23 dwarfing type individuals) segregation (P=0.854, χ²=0.034) for molecular detection. Based on Sanger sequencing results, the linked SNP markers were identified in the position 25 230 425 bp and 27 191 090 bp on Pp06 of peach genome and the locus were on the right side of these two markers. For fine mapping, the parent of this segregation population was resequenced with 66.89X depth and identified the heterozygous SNP to develop SNP markers. Totally, fifteen SNP primers were designed and 12 SNPs (80.0%) were consistent with resequencing data. The dwarfing locus of this type was narrowed on Pp06 between the position 28 712 165 bp and 28 899 661 bp with genetic distances of 0.38 cM and 0.13 cM. The physical region of fine mapping was 277 kb containing 54 known transcripts. Within 28 108 436bp and 29 247 763 bp on Pp06, SNP markers were developed and detected the progeny phenotype of a segregation population of 10-7×96-5-1 with 100% accuracy, constituted of 89 individuals. 【Conclusion】 The dwarfing gene was fine mapped on Pp06 within a physical distance 277 kb. The results of this study will be helpful to clone dwarfing gene and select parents for breeding dwarfing ornamental peach and rootstock varieties.

Effects of Seawater Irrigation on Fruit Quality of Cabernet Sauvignon

HUANG LiPeng, ZHANG XiuYuan, WANG yang, FU YanDong, ZHAI Heng, SHAO XiaoJie

Scientia Agricultura Sinica. 2017, 50(18): 3581-3590. doi:10.3864/j.issn.0578-1752.2017.18.014

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【Objective】This study was carried out to investigate the effects of seawater irrigation on grape fruit quality and to explore the reasonable application of sea water in improving grape quality.【Method】The Cabernet Sauvignon grapes from two bases of Penglai Guobin and COFCO Changcheng were taken as test materials for 4 consecutive years in 2013-2016 in Penglai. Additionally, the different seawater treatments were applied in 2016, including 10% magnetized sea water, and 10% sea water plus fertilizer magnetization, 1 and 2 years 10% water. Some quality parameters were determined, including fruit soluble solids, titratable acid, total phenolics, tannins, anthocyanins, reductive Vitamin C and aroma. 【Result】 The results showed that an experiment of 10% seawater irrigation was conducted at Guobin base, which significantly increased the soluble solids content of Cabernet Sauvignon fruit and decreased titratable acid content, but had no significant effect on the color difference of pericarp in 3 and 4 years. Four seawater irrigation treatments including 10% magnetized water, 10% water fertilizer, 10% water for 1 year and 10% water for 2 years significantly reduced the titratable acid, sugar acid ratio increased at COFCO Changcheng base. Bacterial manure and magnetization seawater significantly increased the content of sugar compared with the control. In contrast, significant difference in fruit sugar components was found among different seawater treatments. Fruit fructose contents from 1- and 2-year seawater irrigation were significantly enhanced by 35.3% and 42.7%, respectively. Glucose increased by 66.7% and 70.7%. The contents of total phenols, anthocyanin content, reductive Vitamin C were increased by seawater treatments. Among them, 4 kinds of seawater treatment significantly increased the Vitamin C content of the fruit by 46.8%, 60.8%, 57.2% and 79.7%, respectively, and adding bacterial fertilizer and magnetized water significantly reduced the content of tannin in fruits. There were 38 kinds of aroma components in Cabernet Sauvignon grape fruits were detected, including alcohols, aldehydes, esters, ketones and other substances. There were 24 aromas in fruits of seawater irrigation treatments and control. Seawater irrigation increased the total content of aroma components of 14 kinds of aroma. Fruit aroma was significantly increased, and total aroma amount was increased by 92.2%, 75%, 25.9%, and 38.8% in the treatments of 10% sea water and bacterial fertilizer, 10% magnetized seawater, one-year 10% seawater and two-year 10% seawater, respectively. Among the main aroma components of (E)-2-Hexen-1-ol, 2- hexenal, (E)-2-hexenal, nonanal and other substances, the highest content of 2- hexenal in the fruits of magnetized seawater and magnetized seawater plus bacterial fertilizer treatments in, increased by 46.9%, 95.6%, 0.5%, 19.3% as compared with the control. 【Conclusion】 It was concluded that 10% seawater irrigation could promote the quality of wine grape, and seawater irrigation increased water soluble solids content and (E)-2-Hexen-1-ol, 2- hexenal aroma components in fruits.

Occurrence, Control and Determination of Patulin Contamination in Fruits and Fruit Products

NIE JiYun

Scientia Agricultura Sinica. 2017, 50(18): 3591-3607. doi:10.3864/j.issn.0578-1752.2017.18.015

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Patulin is a secondary metabolite of polyketide lactone mainly produced by species of Penicillium, Aspergillus, and Byssochylamys. It was found as a contaminant in many fruits and fruit products, the major sources of contamination are apples and apple products, which are also the most important source of patulin in human diet. Patulin has various acute and chronic effects and others at the cellular level. Today, international organizations (Codex Alimentarius Commission and European Union) and many countries across the world have set maximum levels of patulin content in fruit and fruits products. Among the different genera, the most important patulin producer is P. expansum, it can contaminate a number of fruits and fruit products, and produce mycotoxin patulin. P. expansum distributes very extensively, its strains have been isolated from many countries. As a plant pathogen, P. expansum penetrates fruits typically through wounds or injuries produced during harvest and handling, it can also penetrate through stem end, open calyx tube and lenticels of fruits, and infection sites of other primary fruit pathogens. Overmature or long-stored fruits are more susceptible to P. expansum infection. P. expansum is a psychrophile, its growth is quite strong at 0℃. The biosynthetic pathway of patulin consists of approximately 10 steps. It has been clarified that both of the patulin biosynthetic gene cluster from P. expansum and that from A. clavatus composed of the same 15 genes, but their gene sequences differed greatly. The genotoxic and cytotoxic properties of patulin are due to its high reactivity to cellular nucleophiles. Patulin is believed to exert its toxic, chromosome-damaging, and mutagenic activity mainly by covalent binding to cellular nucleophiles, in particular to the thiol groups of proteins and glutathione (GSH). In the major reaction pathways, up to three molecules of glutathione can bind to one molecule patulin. To control the contamination of patulin and the growth of moulds producing it, pre-harvest measures, post-harvest treatments, and storage conditions deserve special attention. The use of chemical fungicides is an important strategy, but the overuse of fungicides will lead to the emergence of fungicide-resistant strains. Because their way of action reduces the risk of resistant population emergence, “low risk fungicides” are more suitable and efficient. Using biocontrol agents are alternative or complementary treatments that permit to decrease fungicide doses or even avoid the use of chemicals. Some stages of manufacturing process (such as washing, sorting and trimming) are highly efficient in reducing the levels of patulin in fruit products. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are classical methods to extract patulin in fruits and fruit products. However, LLE is expensive, time consuming, and unsuitable for the treatment of solid sample. In the last years, some other extraction measures have been studied and developed, including DLLME, IL-DLLME, BS-DLLME, salting out-VALLME, MSPD, and QuEChERS. LC-UV or LC-DAD procedure is routinely used for quantitative determination of patulin, and methods to confirm the presence of patulin usually include more specific detection techniques such as LC-MS/MS and GC-MS/MS. PCR method is a fast and specific method of early detecting the potential patulin producing fungi.

Transcriptome Analysis of Differential Gene Expression Associated with Testis Tissue in Mule Duck and Muscovy Duck

LI Li, MIAO ZhongWei, XIN QingWu, ZHU ZhiMing, ZHANG LinLi, ZHUANG XiaoDong, ZHENG NenZhu

Scientia Agricultura Sinica. 2017, 50(18): 3608-3619. doi:10.3864/j.issn.0578-1752.2017.18.016

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【Objective】 The purpose of this study is to analyze the transcriptome differential gene expression of mule duck and muscovy duck testis, results of the study will lay a theoretical foundation for the further elucidation of the mechanisms of genetic sterility of mule duck. 【Method】 Transcriptome sequencing of testis from mule duck and muscovy duck was performed using the Illumina HiSeq 2500 platform with 2 biological replicates per duck breed, and verified by quantitative real-time PCR (QRT-PCR). 【Result】 After removing sequencing adaptors and the low-quality reads, a total of 43.84 Gb clean reads were obtained, the Q30 base percentage at 91.36% and above, and clean reads were assembled into 193 535 Unigene. The r2 Differential expression analysis showed that 3 597 differentially expressed genes were found between two duck breeds, including 1 194 up regulated genes and 2 403 down-regulated genes. Several genes were related with reproductive function, such as fibroblast growth factor, FGF, protein kinase A (PKA), mitogen-activated protein kinase 7-like, partial (BMK),growth factor receptor-bound protein 2 (GRB2), c-Jun N-terminal kinase (JNK )and tumor necrosis factor receptor superfamily member 6(FAS),and so on. With Gene Ontology (GO) annotation, 382 differentially expressed genes were identified including 97 related annotation genes involving development and reproduction biological process. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differentially expression genes annotated to 50 metabolic pathway, and 17 pathways were enriched significantly, such as calcium signaling pathway, glycerolipid metabolism and MAPK signaling pathway, and gonadotropin releasing hormone (GnRH) signaling pathway and MAPK signaling pathway associated with testis physiology and reproduction activities. Verified by QRT-PCR, the pattern of differential gene expression was consistent with the results of transcriptome sequencing, which showed the sequencing results were reliable. 【Conclusion】 Differentially expressed genes of mule duck and muscovy duck testis were screened by transcriptional analysis, revealed that the GnRH and MAPK signaling pathway play an important role in duck reproductive activities, which provide reliable reference for exploring the differentiation mechanism of reproductive system in mule duck.

Effects of Compound Natural Plant Preparation on Milk Withdrawal and Galactin in Dairy Cows

SHEN LiuHong, XIAO JinBang, WU XiaoFeng, JIANG SiXun, JIANG Tao, DENG JunLiang, ZUO ZhiCai, YU ShuMin, CAO SuiZhong

Scientia Agricultura Sinica. 2017, 50(18): 3620-3630. doi:10.3864/j.issn.0578-1752.2017.18.017

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【Objective】the objective of this study is to explore the impact of compound natural plant preparation on the milk withdrawal and serum insulin(INS), hydrocortisone(HC), growth hormone(GH) and insulin growth factor 1(TGF-1) levels in dairy cows.【Method】A total of 150 Chinese Holstein cows, weighing (582±41) kg, pregnant with 2-4 fetuses, and fed in a large-scale dairy farm in Sichuan Province from January 2016 to June 2016, were used in the present study. Eighty healthy cows with normal milk and udder that in late pregnancy and gave (15.42±0.71) kg milk per day were chosen. The cows were randomly divided into four groups: groups A, B, C and D, with 20 of them in each group. Stop dairy cows’ milk by gradually dry milk method and the day when cows gave less milk was recorded as 0 d. Group A was a control group, did not feed compound natural plant preparation and the groups B, C and D were fed 400g, 500g, 600g compound natural plant preparation, respectively, at 8.00 am from 1 d to the day of dry milk completely. Compound natural plant preparation comprises fructus hordei germinatus, mirabilite, rhizoma cimicifugae, bupleurum, rhizoma cyperi, coix seed, silkworm slough, rhizome atratylodis, scutellaria baicalensis, rhizome anemarrhenae, caulis perllae, semen euryales, schisandra chinensis, dandelion, licorice and so on. The cows’ venous blood was collected from tail on the day 0, 1, 3, 5, 7, 9 and 11, the samples were placed in the centrifuge tubes without anticoagulant at room temperature for 1h. The suspension was centrifuged at 352 × g for 10 min and then put into the refrigerator at -20℃. ELISA was used to evaluate the expression levels of serum INS, HC and IGF-I and the daily milk production was recorded.【Result】Groups A, B, C and D returned milk on the 11 d, 7 d, 5 d and 5 d respectively. The expression levels of serum IGF-1 and HC were declining in all 4 groups and the decline trend of the control group was slower than compound natural plant preparation groups. The expression levels of serum IGF-1, HC and GH were raised after the period of milk withdrawal, the upward trend of the expression levels of serum INS in the control group was slower than compound natural plant preparation groups. From the day 0 to the 1, the expression levels of serum HC, IGF-1 and GH had no significant difference (P＞0.05). From the day 3 to the day7, the expression levels of serum HC, GH and IGF-1 in group A were highly and significantly higher than those of groups B, C and D (P＜0.01), while the expression level of INS in group A was highly significant loser than those of groups B, C and D (P＜0.01). On the day 9, the expression levels of serum HC in groups A and D were significantly higher than that of group C (P＜0.05), the expression levels of serum GH and IGF-1 in groups A and B were highly and significantly lower than those of groups C and D (P＜0.01), the expression levels of serum INS in group A was highly and significantly lower than that of groups B, C and D (P＜0.01). On the day 11, the expression levels of serum HC, GH and IGF-1 in group A were highly significantly lower than those of groups B, C and D (P＜0.01), the expression levels of serum INS in group A was highly significant lower than that of groups B and C (P＜0.01) and significantly lower than that of group D (P＜0.05). The expression levels of serum IGF-1, GH, HC and INS had no significant difference between group C and D during the period of milk withdrawal (P＞0.05). During the period of milk withdrawal, there were significant positive correlations among milk production, HC, GH and IGF-1 (P＜0.01) and there was a significant negative correlation between INS and milk production, HC, GH, IGF-1 (P＜0.01).【Conclusion】 Compound natural plant preparation can help cows milk withdrawal significantly, feeding 500g·d^-1 can shorten the period of milk withdrawal to 5 days can make the best effect on milk withdrawal. The expression levels of serum INS, HC, IGF-1 and milk production had a highly significant correlation. IGF-1 GH and HC are negative regulation factors of milk withdrawal, while INS is a positive regulation factor.

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