Scientia Agricultura Sinica

Genome-Wide Association Analysis on Flag Leaf Width Under Different Nitrogen Levels in Rice

GAO YiHong, YAN JinXiang, TU ZhengJun, LENG YuJia, CHEN Long, HUANG LiChao, DAI LiPing, ZHANG GuangHeng, ZHU Li, HU Jiang, REN DeYong, GUO LongBiao, QIAN Qian, WANG DanYing, ZENG DaLi

Scientia Agricultura Sinica. 2017, 50(14): 2635-2646. doi:10.3864/j.issn.0578-1752.2017.14.001

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【Objective】 The objective of this experiment is to study the genetic mechanism of flag leaf width and its response to different nitrogen fertilizer rates, and provide advantageous germplasm resources and genetic markers for the improvement of nitrogen use efficiency in rice breeding. 【Method】 Based on re-sequencing of 134 rice landraces, a total of 3 356 591 SNPs distributed on the whole genome were identified. Three different nitrogen levels were assigned as the main plot in the split-plot design, and rice landraces are assigned at random to the subplots within each whole plot. Three nitrogen levels including low nitrogen (no nitrogen fertilizer), normal nitrogen (96 kg·hm^-2) and high nitrogen (192 kg·hm^-2) were applied under normal field cultivation, respectively. The EMMAX method was used to analyze the genetic relationship and EIGENSOFT was employed to detect the population structure. The mixed linear model was used to detect the potential genome-wide association between single nucleotide polymorphisms (SNPs) and the flag leaf width performance or response under low, medium and high nitrogen treatments. 【Result】The results showed that the flag leaf width displayed normal distribution in N0, N1 and N2 treatments, respectively. The variation of flag leaf width caused by the varietal differences and the different nitrogen levels, and the significant positive correlation between nitrogen fertilizer and flag leaf width were detected at all nitrogen levels. A total of 14 SNPs on five chromosomes presented significant association with the flag leaf width under three different nitrogen levels. The estimated minimum allele frequency was 0.46 at low nitrogen level, which indicate those loci are widely distributed in the association population. While the estimated minimum allele frequencies were lower at normal nitrogen and high nitrogen levels. One SNP located on chromosome 12 was found both at normal nitrogen and high nitrogen levels. Besides, another SNP on chromosome 12 was also detected at high nitrogen. Its flank contained a candidate gene LOC_Os12g25660, which belongs to the super-family of cytochrome P450. It is a homologous gene of OsBR6ox which was confirmed in regulating the flag leaf width in rice. Based on the variation of leaf width at different nitrogen levels, twenty SNPs and eight SNPs were identified to low nitrogen and high nitrogen response, respectively. Among them, the nitrogen utilization related gene, OsATG7, was associated with high nitrogen response on chromosome 1. 【Conclusion】 In this study, a total of 42 SNPs associated with flag leaf width and response to different nitrogen were identified based on the genome-wide association analysis.

Genotypic Difference of Nitrogen Use Efficiency of Wheat and Correlation Analysis of the Related Characters

XU Qing, XU FuChao, DONG Jing, DONG JianHui, QIN DanDan, LU MengYing, LI MeiFang

Scientia Agricultura Sinica. 2017, 50(14): 2647-2657. doi:10.3864/j.issn.0578-1752.2017.14.002

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【Objective】 The objective of this study was to explore the genetic difference of the nitrogen use efficiency (NUE) of different wheat lines from the middle and lower reaches of Yangtze River and screen out the lines with high NUE, which would provide useful germplasm for wheat high NUE breeding and lay a foundation for uncovering the underlying molecular mechanism. Meanwhile, correlation analysis between NUE and the related traits at different developmental stages of wheat would provide evidences for evaluating the high NUE wheat lines.【Method】The experiment was performed with 118 wheat cultivars mainly from the Yangtze River region in the field conditions by setting low (62.55 pure N kg·hm^-2) and normal (187.5 pure N kg·hm^-2) nitrogen inputs. The genetic variations and correlation analysis were further analyzed by characterizing the traits of dry above-ground mass, tiller number, chlorophyll content at the seedling stage; the dry above-ground mass, plant nitrogen concentration and accumulation at flowering stage; the chlorophyll content at the grain-filling stage; the yield, straw weight, kernel and straw nitrogen concentration and accumulation, spike number, spike length, kernel number per spike, and thousand kernel weight at harvesting stage with individual lines. Finally, classification of the wheat lines according to the respective NUE of each line was undertaken.【Result】The results showed significant genetic variations for most of the tested characters among different wheat lines. Correlation analysis indicated a positive correlation between yield and aboveground dry mass, straw weight, harvest index, spike number of the plant at harvesting stage, dry mass of the plant at the flowering stage as well as the nitrogen accumulation in the kernel and straw of the plant. Meanwhile, except the nitrogen harvest index, a negative correlation was observed between the plant physiological use efficiency and plant straw weight, spike number, kernel and straw nitrogen concentration and accumulation. In addition, according to the yield of the 118 wheat lines under the both nitrogen conditions, the selected wheat lines were categorized into four classes: Efficient-efficient (EE, yield higher than the mean yield of all the lines under both conditions), Inefficient-inefficient (II, yield lower than the mean yield of all the lines under both conditions), and Inefficient-efficient (IE, yield higher than the mean yield of all the lines only under high nitrogen conditions), Efficient-inefficient (EI, yield higher than the mean yield of all the lines only under low nitrogen conditions). The EE type of wheat lines was more sensitive to the nitrogen application. The EI type of wheat lines had significant higher chlorophyll content than the other lines at grain-filling stage, which indicated the critical role of stay-green character of the plant for nitrogen utilization under the low nitrogen conditions.【Conclusion】The selected wheat lines showed significant genotypic variations for their nitrogen use efficiencies under the two nitrogen input conditions. Meanwhile, the different types of wheat lines responded distinctively to the nitrogen application. Among them, the IE type of wheat lines was the most sensitive one to nitrogen application, and suitable for high nitrogen content field, whereas the EE and EI types were more tolerant to the nitrogen starvation, and could be the elite germplasm for high NUE wheat breeding. According to the correlation analysis of yield with different traits, it was found that except the routine characters such as the above-ground dry mass and plant nitrogen accumulation, the spike number could also be used as a trait to evaluate the high nitrogen use efficiency in wheat.

A Spatial Partition Statistical Analysis for Quality and Agronomic Traits of Foxtail Millet Germplasm Resources

CHEN YanQing, CAO YongSheng, CHEN LiNa, FANG Wei

Scientia Agricultura Sinica. 2017, 50(14): 2658-2669. doi:10.3864/j.issn.0578-1752.2017.14.003

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【Objective】 The objective of this paper is to analyze the spatial distribution characteristics of related agronomic traits and quality of millet germplasm resources in China, grasp the overall quality in spatial distribution, improve the macro cognition of millet resources and effective protection and utilization. 【Method】 From the point of view of geographical space, the methods of spatial interpolation and spatial clustering were used to study the spatial distribution rules of the related traits of foxtail millet resources. Firstly, spatial interpolation was made for target traits of foxtail millet resources, partition statistics method was used to analyze national grid and interpolation data and the optimized hot spot analysis method was used to get the spatial partition data. Finally, the high value, random value and low value spatial distribution of foxtail millet related traits were found. 【Result】 The results of the distribution of the foxtail millet resources in the country show that the average of crude protein content of foxtail millet resources is (13.98±1.23)%, the range is 10.47%-17.33%, the coefficient of variation is 8.80%. The average of crude fat content is (4.01±0.38)%, the range is 3.08%-5.47%, the coefficient of variation is 9.48%. The average of grain weight per plant is (10.39±4.13) g, the range is 1.65-29.30 g, the coefficient of variation is 39.75%. The average of growth period is (111.46±10.94) d, the range is 79.15-150.43 d, the coefficient of variation is 9.81%. From the aspect of the cluster distribution areas, the average values of crude protein content in low value area, random value area and high value area are (12.80±0.70) %, (13.98±0.39)% and (15.24±0.42)%, respectively, the amplitude of variations of the three areas, respectively, are 10.47%-14.90%, 12.72%-15.30% and 13.61%-17.33%, the variation coefficients of the three areas, respectively, are 5.47%, 2.79% and 2.76%. The average values of crude fat content in low value area, random value area and high value area are (3.69±0.13) %, (3.99±0.16) % and (4.41±0.26)%, respectively, the amplitude of variations of the three areas, respectively, are 3.11%-4.39%, 3.08%-4.48% and 3.57%-5.47%, the variation coefficients of the three areas, respectively, are 3.52%, 4.01% and 5.89%. The average values of grain weight per plant in low value area, random value area and high value area are (6.49±1.84) g, (10.51±1.49)g and (14.44±2.88)g, respectively, the amplitude of variations of the three areas, respectively, are 1.65-13.38 g, 5.42-16.54 g and 7.63-29.30 g, the variation coefficients of the three areas respectively are 23.73%, 14.18% and 19.94%. The average values of growth period in low value area, random value area and high value area are (99.58±6.64)d, (111.89±2.99)d and (121.17±6.04)d, respectively, the amplitude of variations of the three areas, respectively, are 79.15-116.81d, 99.53-124.44 d and 108.34-150.43 d, the variation coefficients of the three areas respectively are6.67%, 2.67% and 4.98%. 【Conclusion】The internal differences in the high value regions of crude protein content are the minimum and these regions are mainly concentrated in Xinjiang and the northeast of Heilongjiang province. The internal differences of the low value regions are the maximum and they are mainly distributed in the central and Eastern zones. Random value regions are mainly distributed in the areas which located between high value and low value regions. The distribution of crude protein content shows an increasing trend in the two sides. On the contrary, the high value regions of crude fat content own the maximum internal differences, which are mainly concentrated in central zones of China, the low value regions’ internal differences are the minimum, which are mainly distributed in Xinjiang and the northeast of China. The distribution of crude fat content shows a decreasing trend in the two sides and the regions are relatively not regular. The internal differences in random value areas of grain weight per plant are the minimum, and the high value areas are the maximum. Ningxia, Shanxi, Shaanxi and other places belong to the high value areas of grain weight per plant, Heilongjiang, Zhejiang, northern Inner Mongolia, southern Anhui and southwestern parts belong to the low value areas. The internal differences in random value areas of growth period are the minimum, and the low value areas are the maximum. The growth period of foxtail millet in Northeast China, northwest and southwest Henan is relatively long, and Shandong, Hebei and other areas where summer foxtail millet is planted, belong to the short growth period regions.

Advances and Perspectives in Research of Physiological and Molecular Mechanism of Soybean Response to High Temperature Stress

LI JiaJia, ZHENG ShuangYu, SUN GenLou, ZHANG WenMing, WANG XiaoBo, QIU LiJuan

Scientia Agricultura Sinica. 2017, 50(14): 2670-2682. doi:10.3864/j.issn.0578-1752.2017.14.004

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Soybean is a key economic crop, and is an important source of plant protein and oil. In recent years, high temperature (HT) stress caused by global climate change has threatened the key growth period of soybean, and has become one of the main environmental factors which limited the yield and quality of soybean. In order to reveal the molecular mechanism, establish a comprehensive efficiency evaluation system and promoting genetic improvement of soybean resistance to HT stress, the physiological and biochemical basis and molecular regulation mechanism responding to HT stress of soybean were reviewed in this paper. Compared to optimum temperature, the thickness of leaves increased, the stomatal conductance decreased, the membrane permeability increased, cell microstructure and its integrity damaged, the osmoregulation substances (proline, soluble sugar and protein) changed and the key enzyme activities of the antioxidant defense system in soybean have been lost under HT stress, these results will lead to disorders of a series of physiological processes in soybean, which include the photosynthesis, transpiration, respiration and substance content, etc. Besides, HT stress also caused the abnormalities of pollen morphology, the tapetum cytoplasm is not dense, showing vacuolated and autolysis, the pollen vigour and germination rate are significantly decreased, and pollen abortion rate increased, thus resulting in the pod-set rate and seed-set percentage remarkable decrease, and affecting the normal development and protein accumulation of seed and yield formation of soybean, and ultimately leading to lower yields of soybean. Meanwhile, HT stress can also cause some damage to quality traits of seed, and then has an adverse effect on its economic value of soybean. Moreover, normal physiological metabolism is widely affected by HT stress at the level of transcription, translation and metabolism. At present, many transcription factors, proteins and metabolites response to HT stress in soybean have been identified by the method of high-throughput sequencing, however, there is still a wide gap compared with research on rice and Arabidopsis. Currently, the comprehensive stress response indexes based on pollen viability and several physiological parameters have been used to screen HT resistance soybean varieties, but, the method of system identification is still imperfect in soybean production, and thus leading to the slow progress in selection of HT resistant soybean. Up to date, challenges are still existed in preventive measures, the establishment of a comprehensive evaluation system, screening for excellent soybean germplasm related to resistance to HT, and identification of key functional genes resistance to HT stress in soybean. The adverse effects of extreme ecological environment on soybean production will be further aggravated with the uncertainty of global climate change. In the future, the main focuses should be put on the establishment of a comprehensive evaluation technology system, improvement of the identification level of soybean germplasm resources, elucidation of molecular genetic mechanism and excavation of key genes resistance to HT in soybean. Selecting new soybean varieties with resistance to HT stress by both of the conventional breeding and molecular breeding technologies, thus will provide a theoretical basis and technical support for achieving the unification of HT resistance as well as high yield and quality in soybean.

The Relationships Between Root Morphology, N Absorption and Utilization and Grain Yield in Rice with Different N Use Efficiencies

LI Na, YANG ZhiYuan, DAI Zou, SUN YongJian, XU Hui, HE Yan, YAN TianRong, JIANG MingJin, GUO ChangChun, WANG ChunYu, MA Jun

Scientia Agricultura Sinica. 2017, 50(14): 2683-2695. doi:10.3864/j.issn.0578-1752.2017.14.005

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【Objective】The objective of this research is to study the relationships between root morphology, nitrogen (N) absorption and utilization and grain yield in rice with different N use efficiencies, clarify the root morphology characters of high yielding, and put forward the corresponding optimized water-N management practice. 【Method】 In 2013, high N use efficiency (HN) variety Chuannongyou 498 and low N use efficiency (LN) variety Chuanyou 6203 were used as trial materials to conduct a split-plot experiment with different panicle fertilizer ratio. In 2014, a split-split plot experiment was performed with the same varieties, where seedling number per hole was assigned to the split plot, and the proportion of spikelet preserving fertilizer and spikelet promoting fertilizer was assigned to the split-split plot. In 2015, a split-split plot experiment was carried out with water management practice as split plot and N-management practice as split-split plot, where HN variety Dexiang 4103 and LN variety Yixiang 3724 were used as trial materials. Grain yield, root morphology characters, and N absorption and utilization characters, obtained from the above 3 field experiments, were performed multiple collinearity diagnostics through calculating the variance inflation factor. After confirming that there were serious multiple collinearity problems, ridge regression analysis was used as the main data analysis method to investigate the relationships between root morphology characters, N absorption and utilization and grain yield. Based on the recurrence of these relationships from 2013 to 2015, common regularities hidden among these relationships were located. 【Result】 The results were as follows: (1) For HN varieties, the determination coefficients of ridge regression equation between grain yield and N accumulation (NA), N dry matter production efficiency (NDMPE), and dry matter weight (DW) at elongation stage, heading stage, and maturity stage, were in the range of 0.0219-0.3961, 0.0452-0.1379, and 0.0914-0.6694, respectively. For LN varieties, the determination coefficients were in the range of 0.0084-0.6190, 0.1224-0.4341, and 0.0818-0.4881, respectively. The relationship between grain yield and N uptake and utilization was less repeatable during the year, and there was no obvious common regularity. Ridge regression analysis was performed with 11 root morphology characters of total root dry weight, adventitious root number, length, surface area, volume, coarse lateral root length, surface area, volume, and fine lateral root length, surface area, volume as independent variables and NA, NDMPE, and DW as dependent variables, respectively. For HN varieties, the determination coefficients were in the range of 0.0527-0.2728, 0.0653-0.3139, and 0.0714-0.3158, respectively. For LN varieties, the determination coefficients were in the range of 0.0607-0.5040, 0.0555-0.4411, and 0.0724-0.5449, respectively. There were significant differences in the relationships between N absorption and utilization and root morphology among years. From 2013 to 2015, the determination coefficients of ridge regression equation between grain yield and root morphology characters of both HN varieties and LN varieties all exceeded 0.8 (P＜0.001), which showed an common regularity that root morphology at heading stage had a significant effect on grain yield. (2) In the ridge regression equation with root morphology characters as independent variables, and grain yield as dependent variable, the coarse lateral root length of HN varieties obtained the largest standard coefficient, and had the greatest impact on grain yield. However, for the LN varieties, the fine lateral root surface area played the most important role in increasing grain yield. (3) With N rate of 150 kg·hm^-2, the optimal N management practice (panicle fertilizer ratio was 40% and the ratio of spikelet preserving fertilizer to panicle fertilizer reached or exceeded 50%) combined with dry-wet alternate irrigation was beneficial to optimize the root morphology. For the SPAD-diagnosis N management practice, N rate reduced from 150 kg·hm^-2 to 120 kg·hm^-2, conventional irrigation mode was more favorable to root morphology optimization than dry-wet alternate irrigation.【Conclusion】Rice root morphology at heading stage had close relationship with grain yield, and appropriate water-N management practice could optimize rice morphology to increase grain yield. SPAD-diagnosis N management practice coupled with conventional irrigation or optimal N management combined with dry-wet alternate irrigation mode could extend the coarse lateral root length of HN varieties as well as the fine lateral root surface area of LN varieties at the heading stage to increase their grain yields, respectively.

Effect of AMF Inoculation on N Uptake of Interspecific Competition Between Maize and Soybean Growing on the Purple Soil

ZHAO QianXu, SHI Jing, XIA YunSheng, ZHANG NaiMing, NING DongWei, YUE XianRong, YANG HaiHong

Scientia Agricultura Sinica. 2017, 50(14): 2696-2705. doi:10.3864/j.issn.0578-1752.2017.14.006

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【Objective】 Arbuscular mycorrhizal fungus (AMF) is one of the most important soil beneficial microbe in natural ecosystem. It can effectively promote the nutrition absorption of plants and also regulate the interspecific relative competition ability and nutrition competition ratio between plants by redistributing water and nutritive elements between host plants at the intercropping level. To study the effect of AMF inoculation and different intercropping patterns on the interspecific relative competition ability and nitrogen (N) competition ratio between intercropping maize (Zea mays L.) and soybean (Glycine max L.) growing on the purple soil, and provide a scientific basis for the regulation of nutrient resource utilization and competitive effect between crops. 【Method】 A pot experiment with three different intercropping patterns (no separation, nylon net separation, plastic-film separation) and different AMF treatments [no AMF (NM), Glomus etunicatum inoculation (G.e)] was conducted to analyze intercropping plant growth and N nutrition status, which in order to quantify the effects of AMF treatments and intercropping patterns on the interspecific competition ability and N competition ratio between maize and soybean. 【Result】 The results showed that under the same intercropping conditions, maize always had a stronger competitive advantage, soybean showed a competition disadvantage. The interspecific competition ability and N competition ratio between maize and soybean under G.e-no separation treatment both were the highest. Whether AMF inoculation or not, N concentration, N uptake and the root biomass of maize plants were all in the order of no separation pattern nylon net separation pattern plastic-film separation pattern, while soybeans showed an opposite trend. In comparison with NM, AMF inoculation significantly promoted the interspecific competition ability and N competition ratio between maize and soybean, so that shoot and root biomass of maize was increased by 20.48% and 23.33%, respectively, under no separation patterns, and shoots and roots N uptake were also improved by 64.20% and 34.61%, respectively. For soil available N content, G.e-no separation treatment significantly improved N uptake of maize and soybean in intercropping of maize and soybean, and decreased soil available N content, which reduced soil N residue. 【Conclusion】 In conclusion, different competitive ability of maize with soybean was different in all intercropping patterns, but maize was always in a stronger competitive advantage than soybean. AMF inoculation also improved the interspecific competition ability and N competition ratio of maize to soybean significantly. These revealed that AMF played an important ecological significance in regulating resource utilization between different plants and maintaining the diversity of the crop plants in farmland.

Interactive Effects of Nitrogen and Potassium on Photosynthesis Product Distribution and Accumulation of Sweet Potato

WANG ShunYi, LIU Qing, SHI YanXi, LI Huan

Scientia Agricultura Sinica. 2017, 50(14): 2706-2716. doi:10.3864/j.issn.0578-1752.2017.14.007

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【Objective】The objective of this paper is to explore the interactive effects of nitrogen and potassium on photosynthesis product transfer allocation and its physiological mechanism of sweet potato.【Method】The two years field experiments include four treatments: CK, N, K and N+K, ¹³C blade tag on leaves was made on 40 d and 100 d after transplanting. The interactive effects of nitrogen and potassium on ¹³C distribution, photosynthetic characteristics, fluorescence characteristic, SS activities, SPS activities, and yield of sweet potato were studied. 【Result】Compared with N, K, interactions of N and K treatment, in 2014, yield increased by 10.1% and 7.9%, and the interactive effect value was 0.95 t·hm^-2; in 2015, yield increased by 10.7% and 9.3%, and the interactive effect value was 1.35t·hm^-2. Compared with N and K treatments, interactions of N and K treatment improved the functional leaf photosynthesis and chlorophyll fluorescence characteristics, and the accumulation of photosynthetic products increased at two key stages of sweet potato. For example, ΦCO₂increased by 27.1%-39.7%, P_nincreased by 9.1%-20.2%, and ¹³C total accumulation increased by 26.3%-42.2%. At the early stage of growth, nitrogen and potassium interactions treatment significantly increased leaf SS and SPS enzyme activities, their activities were significantly higher than that in root, and significantly improved the ¹³C distribution rate in leaves by 60.7%, promoted photosynthetic products distribution in the source organ. In root rapid expansion period, interactions of N+K treatment significantly increased the SS and SPS enzyme activities of root, which were significantly higher than that of leaf blade, photosynthetic product distributed from the ground to the underground part under the action of pressure in the sink - source organs, nitrogen and potassium interactions increased the ¹³C distribution rate in root by 71.6%. Stepwise regression analysis showed that the carbon metabolism enzyme photosynthetic characteristics and chlorophyll fluorescence characteristics are the key indicators of product distribution of sweet potato(R₁=0.954, R₂=0.912). Path analysis indicated that the largest coefficients affected ¹³C distribution under nitrogen and potassium interaction were Pn, Fv/Fm and SS at the early stage of growth while key coefficients affected ¹³C distribution were Pn, ΦPSⅡ and SPS in root rapid expansion period. 【Conclusion】It was concluded that nitrogen and potassium interaction promoted the accumulation of photosynthetic products in aerial part through increase of Pn, ΦPSⅡ and SS, thus realized the construction of source at the early stage of growth. In root rapid expansion period, N and K interaction mainly improved P_n, ΦPS and SPS and thus promoted the transfer of photosynthetic products from the ground to the underground part and promoted the yield of sweet potato.

Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium Bacillus cereus YUPP-10 Against Verticillium Wilt in Gossypium hirsutum

ZHOU JingLong, FENG ZiLi, FENG HongJie, LI YunQing, YUAN Yuan, LI ZhiFang, WEI Feng, SHI YongQiang, ZHAO LiHong, SUN ZhengXiang, ZHU HeQin, ZHOU Yi

Scientia Agricultura Sinica. 2017, 50(14): 2717-2727. doi:10.3864/j.issn.0578-1752.2017.14.008

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【Objective】Control of Verticillium wilt is a worldwide problem in agriculture and horticulture, screening of effective biocontrol microbial resources has become one of the important ways to control Verticillium wilt. The use of microbial antagonists to control these pathologies fits modern sustainable agriculture criteria. The objective of this study is to screen an efficient antagonistic bacteria, and characterize the biocontrol mechanism of bacteria against Verticillium wilt, thus providing a technical basis for control of Verticillium wilt of cotton with biocontrol bacteria. 【Method】An endophytic bacterium, which can hydrolyze polysaccharides with β-1,4 linkage, was isolated by enrichment medium, which was made of konjac glucomannan as carbon source. The inhibition and prevention of YUPP-10 against V. dahliae and Verticillium wilt were tested. The inhibition rate of YUPP-10 against V. dahliae was assessed using the confront culture method, enclosed chamber test and hanging drop method, cultured microsclerotia with aseptic culture filtrate, and the control effect of cottonon Verticillium wilt was detected by substrate inoculation method in a pot experiment. Induced disease resistance of YUPP-10 in cotton were analysed by embryo and leaves inoculated with V. dahliae, sedimentation of lignin and reactive oxygen species (ROS). In addition, the expression level of defense genes in G. hirsutum leaves were detected by real-time quantitative PCR (qRT-PCR). 【Result】A Bacillus cereus isolate YUPP-10 was screened. The results showed that YUPP-10 and its volatile organic compounds significantly inhibited the colony growth of V. dahliae, with the width of the inhibition zone of YUPP-10 was 0.73 cm after 7 days, and the inhibition rate of volatile organic compounds of YUPP-10 was 77.03% after 10 days. B. cereus YUPP-10 culture filtrate (CF) suppressed V. dahliae spore and microsclerotia germination in a dose-dependent manner. The inhibition rate of CF on V. dahliae spore and microsclerotia germination ranged from 17.22% to 71.25% and 10.69% to 26.62%, respectively. The inhibition rate was 80.60%, when cotton seedlings were pro-inoculated with substrate containing YUPP-10 in a pot experiment. YUPP-10 induced disease resistance, including immature embryo and leaves against V. dahliae, ROS, sedimentation of lignin and some defense genes, such as PAL, POD, PPO, CHI and PR10.【Conclusion】It was concluded that YUPP-10 is an efficient biocontrol agent that protects cotton plant from V. dahliae infection. Our data demonstrate that V. dahliae growth is restricted and the additional signals from YUPP-10 must participate in the regulation of the immune response against V. dahliae. Therefore, the YUPP-10 has a great potential in controlling Verticillium wilt.

Molecular Detection and Identification of Main Viruses on Pepper in Shandong Province

WANG ShaoLi, TAN WeiPing, YANG YuanYuan, DAI HuiJie, SUN XiaoHui, QIAO Ning, ZHU XiaoPing

Scientia Agricultura Sinica. 2017, 50(14): 2728-2738. doi:10.3864/j.issn.0578-1752.2017.14.009

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【Objective】 The objective of this study is to identify the main viruses causing pepper virus diseases in Shandong Province, China.【Method】In 2014-2015, a total of 253 susceptible pepper plant samples were collected from 10 urban areas (Linyi, Rizhao, Qingdao, Yantai, Weifang, Zibo, Jining, Heze, Liaocheng, Dezhou) in Shandong Province. Total RNA and DNA were extracted from the leaves of pepper samples, and were subjected to detect viruses with Geminivirus universal primers (PA/PB), Polerovirus universal primers (POL-F/POL-R) and specific primers of main viruses infecting pepper have been reported by PCR and reverse transcription-PCR (RT-PCR). Each amplified fragment was purified by DNA gel extraction kit, cloned into pMD18-T vector and sequenced. The obtained sequences of PMMoV, PeVYV and BWYV were subjected to BLAST search, and compared with the representative sequences of GenBank which had been registered around the world using the Megalign in DNAStar software. Clustal W method of MEGA 5.05 was used for multiple sequence alignment analysis and the phylogenetic tree were constructed by neighbor-joining (NJ) of MEGA 5.05, with the bootstrap of each branch of phylogenetic tree analyzed 1 000 times. 【Result】The PMMoV and CMV had the highest detection rate, were 61.66% and 60.08%, respectively. TMGMV, BBWV-2, BWYV and TMV were popular with the detection rate of 41.90%, 34.78%, 33.20% and 24.90%, respectively. The detection rates of PCV-2, ToMV, TYLCV, PVY, MABYV, PeVYV, PCV-1, ChiVMV and AMV were 11.86%, 9.88%, 9.09%, 6.72%, 5.53%, 3.56%, 3.16%, 0.79% and 0.40%, respectively. No CaCV, PSV, ChiRSV, TSWV and ToMMV were detected. The complex infection phenomenon showed that co-infections were up to 89.92%, three kinds of viruses mixed infection occurs the highest rate, up to 28.63%, followed by four kinds of viruses mixed infection, up to 25.00%, two kinds of viruses mixed infection, up to 21.77%, five kinds of viruses mixed infection up to 13.31%, and 6 viruses detected in one sample is the extreme case, with the mixed infection rate up to 1.21%. Phylogenetic trees were constructed with partial CP of PMMoV, partial RdRp, CP and MP genes of PeVYV and partial CP and MP genes of BWYV, respectively. PMMoV Shandong isolate SD60 and the China Guizhou isolate had the closest relationship, PeVYV-SDRZ31-1 and Italy isolate IT83 shared the closest relationship, BWYV Shandong isolate SD clustered with Korean isolate LS. 【Conclusion】 Fifteen viruses were detected, among which PMMoV and CMV were the main pathogens. The newly detected viruses are MABYV, PeVYV, BWYV, MABYV, PCV-1 and PCV-2 in pepper in Shandong Province. The occurrence of viral diseases and the primary and secondary relationship of the main types of pepper viruses in Shandong Province were clarified.

Preparation of Acetochlor Microcapsules by Interfacial Polymerization and the Environmental Behavior and Control Efficacy

LAN Yue, HU Yue, WANG Yan, Guo YanZhen, ZHAO HengKe, HE Lin, QIAN Kun

Scientia Agricultura Sinica. 2017, 50(14): 2739-2747. doi:10.3864/j.issn.0578-1752.2017.14.010

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【Objective】 The objective of this study is to prepare acetochlor microcapsules by interfacial polymerization and highlight interrelation among these formulations, biological effect and environmental effect. 【Method】 The acetochlor microcapsules were prepared by interfacial polymerization method with styrene, butyl methacrylate, and diacetone acrylamide in the O/W emulsion. The performances of acetochlor microcapsules such as morphological structure, size distribution and chemical group were characterized by FESEM, DLS, IR and so on. Meanwhile, through study on the release performance of acetochlor microcapsules, the acetochlor residues in soil and the control efficacy on weeds, connection among the release mechanism, environmental behavior and control efficacy were investigated. 【Result】The prepared acetochlor microcapsules are spherical, the average particle size is 1.552 μm, the loading efficiency is 60%, and the content of acetochlor in formulation is 20%. Under laboratory conditions, when pH values kept at pH 7.0, the cumulative release rates of acetochlor were about 50% to 70% on the 5th day, 60% to 90% on the 10th day, and 80% to nearly 100% on the 30th day from 20℃ to 45℃. In the pH range of 5.0 to 9.0, the cumulative release rates of acetochlor were from 60% to 70% on the 5th day, from below 70% to nearly 80% on the 10th day, about 80% on the 30th day, and nearly 100% on the 40th day. The release of acetochlor microcapsules (MC) was accelerated with the increase of temperature and pH. The grassy weeds and total weeds control efficacy were 80.2%-95.4%, and 67.8%-82.1% on the 20th day, 82.2%-92.4%, and 70.2%-80.9% on the 40th day, and 87.0%-97.2%, and 62.6%-91.3% on the 60th day; fresh weight control efficacy were 90.1%-98.0% and 70.3%-88.7% on the 60th day. For the conventional acetochlor concentrated emulsion, the grassy weeds and total weeds control efficacy were 80.4%-90.2%, and 54.8%-76.1% on the 20th day, 83.0%-91.2%, and 61.4%-79.8% on the 40th day, and 81.1%-88.3%, and 71.2%-84.0% on the 60th day, respectively, and fresh weight control efficacy were 80.3%-83.4% and 65.9%-74.0% on the 60th day. Under field conditions, the amount of acetochlor in the soil gradually reduced over time, and the active ingredient amount of acetochlor microcapsules was higher than that of acetochlor emulsifiable concentrate (EC), which extended the control time and ensured acetochlor microcapsules a better control efficacy.【Conclusion】This delivery carrier may be extended to other lipophilic pesticide in the future. At the same time, more pesticide active ingredient for microcapsules than that of EC can improve the weeds control efficacy, and reduce the number of spraying and amount of pesticide for EC, and relieve the pressure on the environment.

Effects of Albic Soil Physical Properties and Rice Yields After Long-Term Straw Incorporation

WANG QiuJu, CHANG BenChao, ZHANG JinSong, HAN DongLai, SUI YuGang, CHEN HaiLong, YANG XingYu, WANG XueDong, JIAO Feng, CHANG BenChao, KEN Araya, LIU Feng

Scientia Agricultura Sinica. 2017, 50(14): 2748-2757. doi:10.3864/j.issn.0578-1752.2017.14.011

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【Objective】This study investigated the effects of long-term rice (Oryza sativa L.) straw incorporation on the physical properties of albic soil and crop yield. 【Method】The experiment included four treatments: (1) CK (no straw incorporation); (2) S (single application of straw); (3) SNPK (straw and N, P, K fertilizer); and (4) NPK (N, P, K fertilizer). In 2005, 2010, and 2015, after the fall harvest, undisturbed soil from different layers was obtained, and soil bulk density, hardness, porosity, three-phase composition and rice yield were analyzed. 【Result】Rice average yield of ten years after SNPK treatment was 275.70% and 133.23% higher than that after CK and S treatment, and was 14.17% higher than that after NPK treatment, rice average yield of SNPK were higher in the 5-10 years than that of the same treatment in the 1-5 years. Long-term straw incorporation improved soil physical properties. After 10 years of SNPK treatment, soil bulk density of the 0-20 cm and 20-30 cm soil layers was reduced by 6.34% and 10.00% than CK, respectively, the trend was degressive than after 5 years; Soil hardness of the 10, 20, and 30 cm soil layers was reduced by 26.87%, 5.68%, and 4.62% after SNPK treatment than before treatment, respectively, the difference was significant between SNPK and CK in 20-30 cm layer, the trend of 10 year was degressive than that of 5 year, but not obvious, other treatment were not obvious degressive trend than CK; and the solid phase ratio of the 0-20, 20-30, and 30-40 cm soil layers was reduced by 10.87%, 10.61%, and 4.67% than CK, respectively. These values were lower than those noted after 5 years of straw incorporation. Furthermore, long-term straw incorporation increased the number of effective pores in the soil, and the difference was greatly significant between treatments and between years. After 10 years of treatment, the proportions of effective pores of the 0-20, 20-30, and 30-40 cm soil layers treated with SNPK were 28.86%, 63.85%, and 23.40% higher, respectively, than those treated with CK. The proportions of effective pores in the 0-20 and 20-30 cm soil layers in the SNPK treatment were 12.55% and 62.96% higher than those in the NPK treatment, the proportions of effective pores in the 0-20, 20-30, 30-40 cm soil layers in the SNPK treatment were 19.68%, 56.52% and 24.46% higher than those in the S treatment, the trend was increased than that after 5 years. Each soil layer showed increasing numbers of effective pores over time with SNPK treatment. Straw incorporation improved the quality of albic soil, however, its effects become apparent over the long term.【Conclusion】Long-term straw incorporation could improve soil physical properties, increased soil effective pore, decreased soil bulk density, and increased rice yield. Good results were obtained from straw incorporation and chemical fertilizer application. The effect of straw incorporation with fertilizer application on soil and rice was obvious than the treatment of single straw incorporation and single fertilizer application.

Yield Based Evaluation on Fertilizer Application and Analysis of Its Reduction Potential in Weibei Dryland Wheat Production

CAO HanBing, WANG ZhaoHui, ZHAO HuBing, MA XiaoLong, SHE Xu, ZHANG Lu, PU YueJian, YANG ZhenZhen, Lü Hui, SHI YuanChao, DU MingYe

Scientia Agricultura Sinica. 2017, 50(14): 2758-2768. doi:10.3864/j.issn.0578-1752.2017.14.012

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【Objective】It is of great significance to clarify the farmers’ nutrient input situation for the realization of stable yield, high yield, and high nutrient use efficiency in dryland wheat production.【Method】A 5-yr long farm survey of 1 261 farmers was carried out to analyze and evaluate their fertilizer applications and the fertilizer reduction potential in Weibei dryland, based on the nutrient requirement determined by the corresponding wheat grain yields and sustainable development of dryland wheat production.【Result】Obtained results showed the farmers’ wheat yields ranged from 750 to 9 000 kg hm^-2, with the average of 4 243 kg·hm^-2, and they were allocated into five groups as: very low (＜2 640 kg·hm^-2), low (2 640-3 780 kg·hm^-2), moderate (3 780-4 920 kg·hm^-2), high (4 920-6 060 kg·hm^-2) and very high (＞6 060 kg·hm^-2), respectively, accounting for 22.0%, 22.2%, 19.3%, 22.8% and 13.6% of the total. Farmers’ nitrogen (N) application rates ranged from 33 to 454 kg N·hm^-2 with an average of 188 kg N·hm^-2, phosphorus (P) ranged from 0 to 435 kg P₂O₅·hm^-2 with an average of 125 kg P₂O₅·hm^-2, and potassium ranged from 0 to 201 kg K₂O·hm^-2 with an average of 19 kg K₂O·hm^-2. However, farmers’ yields showed no significant correlations with the N, P, and K rates, respectively. With the increase of grain yield levels, the proportion of N over application farmers decreased from 97.8% in the very low yield group to 18.0% in very high group, but that of N deficient application farmers increased from 0.7% to 45.9%, correspondingly. Similar to N, the proportion of P over application farmers decreased from 99.3% in very low yield group to 70.9% in very high yield group, and this means P over application was practiced by more than 70.0% of farmers in each yield group. Different from N and P, K deficient application was practiced by more than 60.0% of farmers in each yield group. Therefore, for N, farmers in very low and low yield groups were recommended to reduce 24-144 kg N·hm^-2, 28%-73% from their high or very high N application rates, and farmers in moderate, high and very high yield groups were recommended to reduce 50-181 kg N·hm^-2, 26%-51% of their high or very high N rates and add 38-134 kg N·hm^-2, 41%-345% more to the low or very low N rates. For P, farmers in different yield groups should reduce 7-31 kg P₂O₅·hm^-2, 23%-33% from the high P rates, and reduce 85-118 kg P₂O₅·hm^-2, 61%-85% from the very high P rates. For K, farmers with no or very low K input in different yield groups were suggested to use 13-50 kg K₂O·hm^-2, and add 7-18 kg K₂O·hm^-2, 35%-78% for those with low K rates. 【Conclusion】 Compared with the conventional method, which adopted an uniform fertilization rate as the criterion to evaluate the famers’ fertilizer application with variable yields, the present work proposed a yield based approach. This approach is proved to be suitable for the small scale household farming in China, and enable to objectively and accurately understand the arbitrary and over application of fertilizer, and to provide a scientific basis for the effective regulation of farmers’ fertilizer application.

Experimental Study on Inter-Annual Water Requirement and Water Consumption of Drip Irrigation Maize in North of Xinjiang

ZHAI Chao, ZHOU HePing, ZHAO Jian

Scientia Agricultura Sinica. 2017, 50(14): 2769-2780. doi:10.3864/j.issn.0578-1752.2017.14.013

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【Objective】 The objective of this experiment is to explore the annual water demand of maize, meteorological factors, water productivity, crop coefficient and characteristics of growth and physiological dynamics of maize cultivated by using drip irrigation under membrane in the northern Tianshan area in the south edge of Zhunggar Basin in Xinjiang, and to provide a theoretical basis for adjusting regional crop planting structure and saving water.【Method】At the Xinjiang Irrigation Center Experimental Station, Four different irrigation quotas of 300, 375, 450, and 525 m³·hm^-2 (denoted by T1, T2, T3, T4, respectively) were analyzed in a 3-year irrigation test carried out in 2013-2015. The correlations between the meteorological factors and the water demand of maize were studied by using the correlation analysis method to determine the water requirement, the crop coefficient, the growth index and the physiological index of intercropped maize. Changes of the main meteorological factors were also studied. 【Result】The total water consumption of maize in the interannual period increased with the increase of irrigation amount. The total water consumption in different treatments was 202.69, 243.22, 317.70 and 366.99 mm in 2013, that was 293.81, 372.18, 466.69, and 537.13 mm in 2014, and that was 326.84, 401.31, 490.76 and 569.33 mm in 2015, respectively. The total annual water consumption in 2015 was 5.66%, 4.90%, 7.26% and 10.11% higher than that in 2014, 35.54%, 35.26%, 39.39% and 37.98% higher than that in 2013. In 2014, water consumption was 31.68%, 31.92%, 34.65% and 31.01% higher than that in 2013. The average temperature in 2015 was 2.6%-3.3% higher than that in the previous two years; the effective rainfall was 30.4%-31.2% more than that in the previous two years, and the relative humidity was 11.5%-12.5% higher. The average temperature had a significant effect on the water demand from sowing to grain filling, and the coefficient of determination of linear regression equation was 0.98. The irrigation amount in the growing period was 3 570-6 370 m³·hm^-2, and the yield was 13 061-14 929 m³·hm^-2. The yield of maize was increased first and then decreased with the increase of irrigation amount. Water productivity decreased with the increase of irrigation volume, ranging from 5.92 to 1.75 kg·m^-3. With the irrigation quota increasing, the crop coefficient varied from 0.51 to 1.18. The crop coefficient in the three years was the highest at the grain filling-maturing stages, and its value was 1.01. The sowing-emergence coefficient was the smallest.【Conclusion】 There were significant differences in the total water consumption of maize in the inter-annual period, and the water consumption in the growth period was significant at the stages of jointing-tasseling, tasselling-grouting, and grouting-maturing. There were significant differences in the total water consumption between treatments at different stages, and the water consumption at each growth stage was significantly different between tassel-filling stage, filling stage and maturity stage. The average temperature, effective rainfall and relative humidity of meteorological factors are increasing year by year. Solar radiation, wind speed and average temperature are positively correlated with water demand, and relative humidity is negatively correlated with water requirement. At the jointing-tapping stage, the main meteorological factors are relative humidity and average temperature; At the tassel-grouting stage, the main meteorological factors are the average temperature. The main meteorological factors are solar radiation and wind speed. The average temperature had the greatest influence on water requirement in the maize growing period. The relationship between maize yield and irrigation amount in the whole growth period is quadratic parabola. The whole growth period of maize showed a single-peak change, and the peak appeared at tassel-filling stage. During the growing period, the plant height of maize increased with the increase of irrigation amount, the whole growth period showed a single peak change, and the peak value was at tasseling stage. The peak value of leaf area index appeared at grain filling stage, and chlorophyll peak appeared at tasseling stage.

Cloning and Expression Analysis of Anthocyanin Biosynthesis-Associated DFR and MYB Genes in Calyx of Eggplant (Solanum melongena L.)

WANG HaiZhu, QU HongYun, ZHOU TingTing, XU QiJiang

Scientia Agricultura Sinica. 2017, 50(14): 2781-2792. doi:10.3864/j.issn.0578-1752.2017.14.014

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【Objective】Anthocyanins are the water-soluble secondary metabolites synthesized via the flavonoid pathway responsible for red, violet, and blue in different kinds of plant organs. These pigments are of benefit to human health. Eggplant is rich in anthocyanins. The molecular mechanism of anthocyanin biosynthesis in eggplant calyx is still unclear. The primary objectives of this experiment are to clone DFR and MYB genes in the eggplant, detect their expression at different developmental stages and in calyx with different colors, explore the role of DFR and MYB in anthocyanin synthesis in calyx of eggplant. This work expanded our knowledge about the molecular mechanism of anthocyanin biosynthesis in eggplant peel and calyx. 【Method】Green calyx and purple calyx of eggplants were used as materials. The content of anthocyanins in the calyx was measured by spectrophotometry at different pH. cDNA sequence of the DFR and MYB were isolated and cloned by using RACE techniques. Analyze the conserved domains and characteristics of DFR protein were analyzed. Phylogenetic analysis of homologous protein sequences of other plants and DFR was conducted, the phylogenetic tree was constructed to further analyze and identify genes. To predict its secondary structure, SPOMA was used on-line which provided by the ExPAsy website. The bioinformatics analysis of MYB was the same as DFR. Expression of DFR and MYB genes in calyx with different colors were investigated through quantitative real-time PCR.【Result】In this study, two full-length cDNA sequences of DFR were successfully cloned and denoted as ouSmDFR and dongSmDFR (GenBank accession Nos KX224250 and KX224251). ouSmDFR is 1 285 bp in cDNA length with an open reading frame of 858 bp corresponding to 285 amino acid residues. dongSmDFR is 1 249 bp in cDNA length with an open reading frame of 864 bp, which encoding 287 amino acids. Two full-length cDNA sequences of MYB gene were cloned and denoted as ouSmMYB and dongSmMYB (GenBank accession Nos KX224253 and KX224254). The corresponding cDNAs are 969 bp and 959 bp in length and the deduced proteins contained 153 amino acids, respectively. Results of secondary structure analysis exhibited that α-Helix and random coil were primary secondary structural components of the two DFR genes and two MYB genes. Sequence analysis showed that ouSmDFR and dongSmDFR have a NADPH-binding domain and substrate specific binding domain. ouSmMYB and dongSmMYB belonged to the R2R3-MYB transcription factors, which have R2 and R3 conserved domains and a bHLH binding domain. Phylogenetic tree analysis indicated that ouSmDFR and dongSmDFR have a close relation with StDFR and SlDFR, ouSmMYB and dongSmMYB have the closest genetic relationship with EsMYB. Results of total anthocyanins measurement showed that high contents of anthocyanins were detected in purple calyx and increased gradually, while no anthocyanins were detected in green calyx. The qRT-PCR analysis indicated that the expression of DFR and MYB genes in the purple calyx were much higher than that in the green calyx. Moreover, from early budding to full flowering,the expression of dongSmDFR and dongSmMYB increased gradually. However, ouSmDFR and ouSmMYB almost had no change, which were consistented with the color variation of calyx.【Conclusion】ouSmDFR and dongSmDFR belong to the NADB-Rossmann superfamily, ouSmMYB and dongSmMYB are typical R2R3-MYB transcription factors. The expression of DFR and MYB in the purple calyx is significantly higher than that in the green calyx. Therefore, it is speculated that the DFR and MYB genes play a role in the color of eggplants’ calyx. Furthermore, DFR and MYB are involved in the process of anthocyanin biosynthesis.

Regeneration of New Germplasms Using Anther Culture of Apple Cultivar ‘Gala’

WEN Xin, DENG Shu, ZHANG ChunFen, HOU LiYuan, SHI JiangPeng, NIE YuanJun, XIAO Rong, QIN YongJun, CAO QiuFen

Scientia Agricultura Sinica. 2017, 50(14): 2793-2806. doi:10.3864/j.issn.0578-1752.2017.14.015

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【Objective】Anther culture is one of the most effective techniques to create new germplasms in modern breeding. Apple is one of the most highly genetically heterozygous and self-incompatible fruit tree. Among the current major apple germplasms, ‘Gala’ cultivar shows traits of early maturity, high yield and enhanced anti-biotic stress, thus is a very important genetic resource for apple breeding programs. Haploid breeding has already been employed to regenerate new germplasm in apple breeding research. In this study, the plantlets were regenerated through embryogenesis during “gala” cultivar anther culture lines to enrich parental apple breeding germplasms. 【Method】The anther culture was used to regenerate gala plants. The ploidy level of regenerated plantlets was determined using flow-cytometry. Subsequently, using selected SSR (HIDRAS) markers, the genotypes of regenerated lines were characterized by gel electrophoresis as well as fluorescent capillary electrophoresis. The anther of ‘Gala’ cultivar collected at early stage were firstly treated at low temperature and then cultured in vitro through embryos induction and differentiation phases and the regenerated plantlets were obtained. The ploidy levels of regenerated plants were analyzed using FACS flow cytometry. To identify the genotype of the plantlets, the DNA was isolated from leaves and then subjected to PCR amplification based on 80 SSR primers selected from the apple HIDRAS database. The homozygous genotypes were determined using both gel electrophoresis and fluorescence capillary electrophoresis. After transplantation, the morphological characteristics of each regenerated plantlet were analyzed.【Result】In the past 3 years, by using the previously optimized anther culture technology, a total of 74 200 ‘Gala’ anthers were inoculated with embryo induction of 0.7% (contaminated anthers over 50 000), resulted in 386 embryos. With differentiation culture, 64 regenerated plantlets survived with regeneration rate of 16.6%. After root induction phase, finally 30 regenerated lines including 28 diploids, 1 haploid and 1 tetraploid were obtained. The PAGE analysis showed that all the regenerated lines were originated from haploid pollen. For genotyping these regenerated plantlets, 17 of 80 SSR markers were further selected (the remaining SSR markers were not polymorphic thus cannot be used for the genotyping) for PCR amplification. Results showed that the panel of 17 SSRs each located in 17 linkage groups, respectively, well distinguished genotypes of the 30 individual regenerated lines. Subsequent morphological observation at the time of 60 days subculture showed that the height of plantlets and morphology of the leaves varied largely. In addition, variations regarding variant ploidy levels were also observed: Gala 5 line was relatively high with wider leaf base, while Gala 7 line showed smaller and thicker leaves and Gala 18 line showed smaller leaves and less amount of leaves. Moreover, diploid plantlets showed a trend of weaker growth than that of the parental ‘Gala’ but stronger than that of haploid and homozygous tetraploid.【Conclusion】By using the technique of anther culture, a set of apple germplasm was successfully obtained and a SSR marker identification system was established. More importantly, the regenerated lines have greatly enriched the apple germplasms and will lay a foundation for apple haploid breeding.

Factor Analysis and Cluster Analysis of Mineral Elements Contents in Different Apple Varieties

KUANG LiXue, NIE JiYun, LI ZhiXia, GUAN DiKai, WU YongLong, YAN Zhen, CHENG Yang

Scientia Agricultura Sinica. 2017, 50(14): 2807-2815. doi:10.3864/j.issn.0578-1752.2017.14.016

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【Objective】 Studies on mineral elements characteristics in different apple varieties were conducted to provide a scientific basis for apple nutrition evaluation, apple consumption and parent selection. 【Method】 Seven indexes including K, P, Mg, Ca, Zn, Cu and Mn in 125 apple cultivars were determined and the contents were analyzed by factor analysis and cluster analysis.【Result】 The average contents of 7 mineral elements were followed by the order of K (1112.72 mg·kg^-1)＞P (119.59 mg·kg^-1)＞Mg (65.69 mg·kg^-1)＞Ca (56.96 mg·kg^-1)＞Zn (0.69 mg·kg^-1)＞Cu (0.66 mg·kg^-1)＞Mn (0.63 mg·kg^-1). The coefficient of variation was varied from 19.4% (Mg) to 43.9% (Cu). There were significant differences among K, P, Mg and Ca (P＜0.05), and no significant differences among Zn, Cu and Mn. All of the 7 mineral element contents obeyed the normal distribution by K-S test. The result of factor analysis showed that Cu, K, Mg, P and Mn were the characteristic elements of apples and the cumulative variance contribution rate of the first four components was 86.30%. The F1 (factor 1) represented Cu, K, Mg, P and the top three apple cultivars for F1 score were ‘Qiujin’, ‘May’ and ‘Guoqing’. The F2 represented Mn and the top three apple cultivars for F2 score were ‘Stark Spur Delicious’, ‘Chunxiang’ and ‘Yellow Transparent’. The F3 represented Zn and the top three apple cultivars for F3 score were ‘Yan shan hong’, ‘Szampion’ and ‘4-23’. The F4 represented Ca and the top three apple cultivars for F4 score were ‘Black Ben Davis’, ‘Fujin’ and ‘Pacific Rose’. The top five apple cultivars for synthesis scores were followed by the order of ‘Qiujin’, ‘May’, ‘New Ralls’, ‘Beidou’ and ‘Changhong’. The 125 apple samples were divided into 5 groups by cluster analysis. The first group included 9 samples in which the Ca content was very high, the second group included 28 samples in which the Cu content was very high, the third group included 23 samples in which the Zn content was very high, the fourth group included 44 samples in which the contents of Ca, Cu, K, Mg, P, Zn were relatively low and the fifth group included 21 samples in which the contents of K, Mg, Mn and P were high and the Zn content was low. 【Conclusion】 There were very significant positive correlations among K, Mg, Cu and P, and all the correlation coefficients were greater than 0.5. Cu, K, Mg, P and Mn were the characteristic elements of apples. The compositions of 7 mineral elements were different among different varieties. The 125 apple samples were divided into 5 types including high Ca variety, high Cu variety, high Zn variety, the variety with high Cu, K, Mg, P and Mn contents and the variety with low mineral element contents.

Expression Patterns and Correlation of Wnts and TET1 Genes in Early Fetal Tissues of Dazu Black Goat

TAN Qiang, LUO NanJian, ZHANG YanLi, AN BingXing，CHEN QiuYang, ZHAO YongJu

Scientia Agricultura Sinica. 2017, 50(14): 2816-2825. doi:10.3864/j.issn.0578-1752.2017.14.017

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【Objective】The aim of the research was to quantitate the relative expression level of TET1, Wnt and DKK genes during early fetus development in goat, analyze the correlation between TET1 and Wnts genes in further. 【Method】Twelve healthy Dazu black goats copulated with the same ram by wild pairing while these goats with spontaneous estrus. Caesarean section was used to obtain fetus that pregnancy on days 20, 25, 30, 60 and 90, respectively, the growth indexes of the fetus (fetal weight, fetal length) were tested, and the tissue samples of 60d and 90d (fetal heart, liver, lung, kidney, brain and skin) were collected. Real-time (RT-PCR) was used to detect the relative expression level of TET1 gene, DKK family genes (DKK1, DKK2, DKK3) and Wnt family genes (Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt5b, Wnt7b, Wnt16). SPSS software was used to analyze the correlation between TET1 and Wnt signaling pathway genes (P＜0.05) at different stages of fetal development. 【Result】 According to the statistics, there were significant changes in fetal development in early pregnancy after 60d. The results showed that the expression of TET1 gene increased with the development of gestational days. All Wnt family genes were detected in the fetus (Wnt2, -2b, -4, -5a, -5b, -7b, -16). Among these changes of fetal development, Wnt2 and Wnt7b expression increase with fetal development gradually; Wnt2b, Wnt5a, Wnt5b, and Wnt7b genes has a significant highly expression on pregnancy 30d (P＜0.05); the expression of Wnt4 was significantly in fetus on pregnancy 20d (P＜0.05); Wnt16 gene was significantly in fetus on pregnancy 25d (P＜0.05). The expression level of DKK family genes showed that DKK1 was detected in the early pregnancy of fetal development, and DKK2/3 performed a low expression level in the early pregnancy, whereas increased in later stage. The expression level of TET1 in liver, lung, kidney and brain in 90d fetus was higher than 60d fetal tissue, there was a significantly expression in liver (P＜0.05). Interestingly, Wnt2 had a highly significant expression level in all tissues of fetus on pregnancy 60 and 90d, especially in lung (P＜0.01); Wnt16 had a highly expression in fetal skin (P＜0.05); Wnt5a and Wnt7b had a significantly expression level in kidney (P＜0.05), other Wnt genes of Wnt family were detected in fetal tissues as well. Correlation analysis showed that the fetal growth indexes (fetal weight, body length) were positively correlated to changes in the expression of TET1 (P＜0.01); TET1 in early fetal development and the expression of Wnt2, Wnt7b and Wnt16 gene showed a positive correlation, and negatively correlated with Wnt2b, Wnt4, Wnt5a and Wnt5b, which was negatively correlated with Wnt5b (P＜0.05), and significantly and positively correlated with Wnt7b (P＜0.01). There was also a correlation between Wnt pathway genes, Wnt2 and Wnt4 showed a significant negative correlation (P＜0.01). Wnt2 and Wnt7b, Wnt2b and Wnt5a, Wnt5b, Wnt5a and Wnt5b showed a significant positive correlation (P＜0.01). Wnt4 was positively correlated with Wnt5a (P＜0.05). 【Conclusion】 The dynamic expression patterns of TET1 and Wnt genes in early fetal development of goat, and correlation of these genes were obtained from this study which fill the blank of those genes in this research field. The article revealed TET1 and Wnts regulate the embryonic development and the formation of tissues by a dynamic pattern. TET1appeared a positively correlation with some Wnt genes while negatively with others. It is also showed interactive in Wnt signaling pathway genes. The data obtained from the research will provide a reference for molecular mechanism research of TET1 and Wnts.

Analysis of Differential Expression Genes Between Different Myofiber Types in Chicken Skeletal Muscle Based on Gene Expression Microarray

SHU JingTing, JI GaiGe, SHAN YanJu, ZHANG Ming, XIAO Qin, TU YunJie, SHENG ZhongWei, ZHANG Di, ZOU JianMin

Scientia Agricultura Sinica. 2017, 50(14): 2826-2836. doi:10.3864/j.issn.0578-1752.2017.14.018

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【Objective】 It has been well documented that myofiber type composition can profoundly influence postnatal muscle growth and meat quality, a higher content of red (oxidative) fibers in muscles exhibits excellent meat quality than muscles contain a high content of white (glycolic) fibers. However, the molecular processes that govern the expression of specific fiber type and the phenotypic characteristics of skeletal muscle remains unclear. In the present study, the transcriptional differential analysis between red and white myofiber types was firstly studied in Qingyuan partridge chickens in order to identify key factors that regulates chicken myofiber composition and transition. 【Method】A global gene expression profiling investigation was conducted to identify differentially expressed genes between red (soleus) and white (extensor digitorum longus) muscle of Qingyuan partridge chickens using the Agilent Chicken Gene Expression Chip. qRT-PCR assays were used to validate the microarray hybridization results, and lentivirural plasmids pack-aging system was used to establish a lentiviral vector for RNA interference of specific targeting chicken PPARGC1A gene to study the function of the target gene. 【Result】 A total of 1224 genes with at least 2-fold differences were identified at the P <0.05 significance level (P <0.05, FC ≥ 2). Compared with the expression of transcripts in EDL, a set of 654 transcripts belonged to the up-regulated group, and another set of 570 transcripts belonged to the down-regulated group in SOL. Although fold changes of the selected genes were not exactly the same between real-time PCR assay and microarray assay, the expression tendencies were highly consistent, suggesting that the data from microarray assay in this study was reliable. A total of 74 significantly different GO terms (P value＜0.05) were obtained. These terms were categorized into one of three categories: biological process, molecular function and cellular component. KEGG pathway analysis revealed that some well known pathways affecting muscle fiber transition, muscle development and lipid metabolism were enriched in both types of muscles. The results of the GO, KEGG pathway and gene coexpression network analysis indicated that PRKAG3, ATP2A2 and PPARGC1A might be the key genes related to chicken muscle fiber characteristics, and PPARGC1A was selected for the further functional analysis. Genes involved in the calcium signaling such as PPP3CA and MEF2C as well as MyHC SM isoform were significantly down-regulated, while MyHC FRM isoform was significantly up-regulated by PPARGC1A knockdown after shRNA interference. 【Conclusion】The analysis presented the gene expression profiles and identified DEGs that may be related to the phenotypic differences between red (SOL) and white (EDL) muscles in chickens. Further shRNA analysis demonstrated that PPARGC1A might play an important role in chicken myofiber composition and can co-activate the transcriptional activity of calcium signaling genes. Results of this study will provide new clues to understand the molecular basis of chicken myofiber composition and transition, and also will provide a theoretical basis for improving and controlling meat quality of chickens.

Expression, Purification, Structure and Activity Analysis of Alkaline Phosphatase of Bombyx mori

HE HuaWei, WANG YeJing, HOU Li, LI Yu, WEI ShuGuang, ZHAO Peng, JIANG WenChao, ZHAO Ping

Scientia Agricultura Sinica. 2017, 50(14): 2837-2850. doi:10.3864/j.issn.0578-1752.2017.14.019

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【Objective】Alkaline phosphatase (ALP) is the key enzyme in the metabolism of phosphoric acid in vivo. The properties of ALP in different species are closely related to their physiological functions. The characterization of the property and structure of Bombyx mori ALP (BmALP) will facilitate to reveal the physiological function and regulation mechanism of ALP in insects.【Method】The total RNA was extracted by Trizol method from the midgut of B. mori larvae on day 3 of the 5th instar, and then cDNA was synthesized with the extracted total RNA as the template by reverse transcription. The upstream and downstream primers were designed by Primer Premier 6.0 software, and BmALP was cloned with the synthesized cDNA as the template by PCR. BmALP and different expression vectors were double digested, respectively, then ligated and transformed into the expression strain. The recombinant protein was expressed by Escherichia coli. The expressions of different expression vectors in the supernatant were compared, and the vector with the best expression of soluble recombinant protein was chosen. The recombinant protein was expressed in large scale using Origami B (DE3) cells, and digested with Prescission protease at 4℃ for 20 h followed by the purification via Ni-NTA affinity chromatography. Then the fusion His-Trx tag was removed using Ni-NTA affinity column again. The molecular weight and the state of BmALP in solution were analyzed by gel filtration chromatography. The secondary structure of BmALP and the effects of temperature on its structure were studied by circular dichroism spectroscopy. The optimum pH, optimum temperature, Km, structural stability and the effect of metal ions on the activity of BmALP were studied by the activity assay.【Result】 The total RNA was extracted from the midgut of B. mori and cDNA was synthesized by reverse transcription. BmALP was successfully cloned with the cDNA as the template. The expression vectors of BmALP with pSKB2, ppSUMO and pET32M.3C were constructed, respectively. The expression analysis showed that the pET32M.3C vector facilitated the expression of the recombinant fusion protein His-Trx-BmALP in the form of a soluble protein in the supernatant of cell lysate. Then the recombinant BmALP was expressed in large scale with the pET32M.3C vector. The soluble recombinant His-Trx-BmALP was purified via Ni-NTA affinity chromatography. After the digestion of His-Trx-BmALP by Prescission protease, the fusion His-Trx tag was removed by Ni-NTA affinity column. Gel filtration analysis showed that BmALP formed a stable dimer in solution. Circular dichroism spectroscopy showed BmALP contained α-helical structure, and its content decreased with increasing temperature. Enzymatic activity analysis revealed that the optimum pH and temperature of BmALP were 11.0 and 45℃, respectively. The Km of BmALP was measured to be 1.40 mmol·L^-1. After 2 h incubation at 10℃, BmALP had the highest residual activity, and the residual activity was completely lost after incubation at 35℃ for 2 h. Mg²⁺ and Zn²⁺ promoted the catalytic reaction of BmALP with the optimal concentration of 40 and 5 mmol·L^-1, respectively. Cu²⁺ activated BmALP activity within 20 mmol·L^-1, and the optimal concentration was 10 mmol·L^-1, however, Cu²⁺ inhibited the activity of BmALP while its concentration was higher than 20 mmol·L^-1.【Conclusion】BmALP was cloned. BmALP protein was expressed and purified and its structure and properties were analyzed. The results of this study provided a basis for further study of its structure and function.

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