Scientia Agricultura Sinica

Genetic Analysis and Fine Mapping of a Novel Rolled Leaf Gene in Rice

LIU Chen, KONG Wei-yi, YOU Shi-min, ZHONG Xiu-juan, JIANG Ling, ZHAO Zhi-gang, WAN Jian-min

Scientia Agricultura Sinica. 2015, 48(13): 2487-2496. doi:10.3864/j.issn.0578-1752.2015.13.001

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【Objective】Rice leaf is not only a vital site for photosynthesis, but also an important factor for ideal plant architecture in rice. This study is useful in map-based cloning of target gene and marker-assisted transferring rolled gene in rice breeding programs. 【Method】A mutant rl16(t) (rolled leaf 16) with rolled leaves was derived from the Indica type rice 9311(wild -type WT) via the radiation of ⁶⁰Co-γ. Then, it was planted for several generations to test the stability of mutant phenotype in the field. At heading stage, rl16(t) and WT were randomly selected 10 strains to measure the main agronomic traits including leaf rolling index, respectively.The same part of leaf of rl16(t) and WT was taken and fixed by FAA, then dehydrated in series ethanol and paraffin embedding. After that, the fixed leaf was incised to 10 microns slices and these slices were stained by sarranine. Finally, the bulliform cells were observed, the number of them was counted and their area was measured under the microscope. Chlorophyll content of rl16(t) and WT were tested at tilling stage. In addition, the leaf phenotype of the F₁ plants and F₂ population were investigated, and the mode of rolled gene inheritance was analyzed by chi-square test. To fine-map the Rl16(t), an F₂population was generated by crossing between the rl16(t) mutant and a Japonica type rice Dianjingyou. Target gene was limited by simple sequence repeat (SSR) and InDel markers. Three known domain structure genes in the mapped region were analyzed by gene quantitative expression. 【Result】Compared with wild-type, the mutant had not only typically adaxially rolled leaves, but also decreased height and panicle length, as well as the seed setting percentage. The whole plant of rl16(t) appeared leaf longitudinally rolled into the approximate tubular phenotype at seedling stage (three leaves and one new), then kept it at the rest of time. On the contrary, WT had flat leaves in the entire life. Paraffin section of flag leaf indicated that the number and area of bulliform cell of rl16(t) were lower than that of WT. The number of bulliform cell in WT was (385.1±43.6) cells/mm², while that in the rl16(t) was(1059.5±254.4) cells/mm². However, for the bulliform cell, there were no significant and obvious changes of rl16(t) and WT. The carotenoid content of rl16(t) was lower than that of WT, but the chlorophyll a, chlorophyll b and total chlorophyll content were significantly higher than that of WT. All of the F₁ hybrid between rl16(t) and WT indicated flat leaves, meanwhile, in an F₂ population, rolled and flat leaf plants segregated in a 3﹕1 ratio (97 rolled versus 326 flat leaves, chi-square = 0.86＜chi-square 0.05 = 3.84). Cytological analysis indicated that the rolled leaf phenotype might be caused by the change of number and size of bulliform cells. Genetic analysis indicated that the rolled leaf characters were controlled by a recessive nuclear gene. Using SSR markers, Rl16(t) was initially mapped in the region between the RM23769 and RM23916 on the long arm of chromosome 9. Furthermore, with enlarged population and more developed InDel markers, Rl16(t) was finally delimited to a 51 kb region governed by the InDel marker DF70 and SSR marker RM23818. Three known structure coding genes (LOC_Os09g09320, LOC_Os09g09360, LOC_Os09g09370) were predicted in the limited interval. The expression of LOC_Os09g09320 and LOC_Os09g09370 in rl16(t) showed no obvious differences with that of WT, but the expression of LOC_Os09g09360 in rl16(t) was only the half of WT.As for quantitative expression analysis of the eight genes that contributed to the rolled leaf, seven of them (SLL1, ROC5, RL14, SRL1, ACL1, NRL1, NAL7)had a decline in the rl16(t) mutant, while OSZHD1 appeared a rise.【Conclusion】The rolled leaf of rl16(t) was due to reduced number and area of bulliform cells. In addition, thisgene would be a putative novel rolled leaf gene. As the result of quantitative gene expression, LOC_Os09g09360 may be the target genes.

Comprehensive Evaluation and Screening Identification Indexes of Shade Tolerance at Seedling in Soybean

WU Xiao-ling, LIANG Hai-yuan, YANG Feng, LIU Wei-guo, SHE Yue-hui, YANG Wen-yu

Scientia Agricultura Sinica. 2015, 48(13): 2497-2507. doi:10.3864/j.issn.0578-1752.2015.13.002

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【Objective】The relay-strip intercropping can expand planting area, increase production, and alleviate the domestic imbalance between supply and demand in soybean industry. However, soybean is suffered from shade stress by taller maize at seedling stage in relay-strip intercropping system. Under the shade stress, soybean morphogenesis and some physiological and biochemical characters changed significantly, which induced yield and quality reduction. The aims of this work were to study the shade tolerance of different genotypes, and provide the simple, effective, and suitable evaluation indicators of shade tolerance, and identify shade tolerance at seedling stage, and provide fine basic materials in soybean.【Method】After 20 days of emergence, twenty four morphological, biomass and physiological indexes correlating with shade tolerance of 19 soybean materials were determined under normal light (1 000 µmol·m^-2·s^-2) and weak light (400 µmol·m^-2·s^-2), such as plant hight (PH), stem diameter (SD), nodes of main stem (HN), hypocotyl length (HL), leaf area (LA), leaf thickness (LT), dry weight (DW), chlorophyⅡ a content (Chla), net photosynthetic rate (Pn), transpiration rate (Tr), initial fluorescence (Fo′), maximum fluorescence in light (Fm′) and so on. Shade tolerance coefficient (STC) of all indexes from both treatments were analyzed by using principal component analysis, membership function method, hierarchical cluster analysis and stepwise regression analysis. 【Result】 Compared with the control, the changing trend and range of morphological and physiological index were different. Six independent comprehensive components were obtained from 24 single indexes by principal component analysis, which reflected 87.65% information of all traits information. The contribution ratio of 6 principal component factors were 30.50%, 21.24%, 12.00%, 10.71%, 8.02% and 5.18%, respectively. The comprehensive shade tolerance values (D) were calculated from the membership function methods, and 19 soybean materials were divided into three shade tolerance types by cluster analysis, 2 materials were high shade tolerance type (class Ⅰ), 5 materials were medium shade tolerance type (class Ⅱ), and 12 materials were shade intolerance type (class Ⅲ). A mathematical evaluation model for soybean shade tolerance was established by stepwise regression equation: D=-0.301+0.419LDW+0.169Gs+0.031PH+0.255Fm(R²=0.959). The predictive values (VP) were basically consistent with the comprehensive shade tolerance value (D), which indicated that the use of the four indexes including leaf dry weight (LDW), stomatal conductance (Gs), plant height (PH) and maximum fluorescence in dark (Fm) to predict the shade tolerance in soybean at seedling stage is feasible. The LDW, Gs and PHof high shade tolerance type were intermediate among three types. The PH and STC of the four indexes in high shade tolerance type were the highest.【Conclusion】A comprehensive evaluation system established by multivariate statistics was used to identify shade tolerance at soybean seedling stage. In same condition, four indicators (LDW, Gs, PH, and Fm) can be used for rapid identification and prediction of soybean materials.

Tobacco Rapid Backcross Improvement Mediated by Arabidopsis Flowering Gene FT

CHANG Ai-xia, GUO Li-jie, LIU Dan, LUO Cheng-gang, WANG Lin-song, FENG Quan-fu, WANG Lan

Scientia Agricultura Sinica. 2015, 48(13): 2508-2517. doi:10.3864/j.issn.0578-1752.2015.13.003

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【Objective】In order to shorten the conventional back-cross breeding cycle, a rapid back-cross way was studied to improve crops using tobacco as a model plant.【Method】Using flue-cured tobacco variety FC8, which is immune to TMV, as the resistant donor parent, and flue-cured tobacco variety Zhongyan 100, which is susceptible to TMV but with good comprehensive properties, as the receptor parent, the following experiments were conducted. First, the Arabidopsis thaliana FT gene was cloned and connected to plant expression vector P22, which was then transferred into Agrobacterium, and leaf-disc method was adopted for gene transformation to create FC8 positive plants containing FT. These positive plants were hybridized with Zhongyan 100 respectively, and according to the separation ratio between early-flowering and non early-flowering plants in F₁ progeny, the positive plants containing single FT gene were identified. Second, the early-flowering plants were selected from the target F₁ progeny to backcross with Zhongyan 100. According to the separation ratio of FT and N gene (TMV resistance gene) marker in BC₁progeny, the positive plants containing single FT gene and in which FT gene was not linked to N gene were identified. Next, continuous backcross with Zhongyan 100 was conducted using the specific positive plants, and plants with FT and N gene marker at each earlier backcross generation were selected. In the final generation, the plants containing N gene marker but no FT marker were selected, and identified whether they contained genetically modified components such as FT, Ubi promoter, Nos terminator and Hyg selection marker. Plants free of those components were continuously self-crossed to make Ngene isozygoty. 【Result】A total of 8 positive plants containing FT were obtained by transgenic technology, one of which was identified to contain single FT gene, and which FT gene was not linked to TMV resistance gene. Using it as the early-flowering TMV resistance donor parent and Zhongyan100 as the recurrent parent, Zhongyan 100 was improved through backcross. The BC₄F₁ progeny were obtained in more than one year, and the time was shortened by 450-500 d compared with conventional backcross breeding. It was found that the transgenic FT gene could be expressed stably in hybridization and backcross progenies. The plants containing FT gene budded at the 50-60 d in the age of seedling, and when they budded, they had 3-4 leaves and the plant height was about 35 cm on average, while the height of non early-flowering plants was about 27 cm and still at the vegetative growth stage. The normal plants budded at the 130-140 d, and when they budded, they had 18-20 leaves and the plant height was about 120 cm on average. Compared with the normal plants, the growth period of early-flowering plants was shortened by 70-90 d, which means that half time of the backcross breeding cycle can be shortened, and the new improved BC₁, BC₂, BC₃germplasms with TMV resistance were also obtained. 【Conclusion】With the FT gene-mediated early flowering and TMV gene markers, the backcross breeding cycle can be effectively shortened and the tobacco TMV resistance can be improved rapidly. Although it is necessary to use transgenic technology to accelerate the generation process, the transgenic gene can be separated out successfully, and the available innovation germplasms containing no genetically modified elements were obtained.

Quantitative Inversion of Key Seedling Condition Parameters in Winter Wheat at Booting Stage Using Remote Sensing Based on HJ-CCD Images

TAN Chang-wei, YANG Xin, LUO Ming, MA Chang, YAN Xiang, CHEN Ting-ting

Scientia Agricultura Sinica. 2015, 48(13): 2518-2527. doi:10.3864/j.issn.0578-1752.2015.13.004

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【Objective】Application of satellite remote sensing data can timely get field planting crops ‘planar’ growth information, accurately reflect the situation and trend of crop seedling condition, serve the yield forecast and actual production. The purpose of this research was to deepen the mechanism and methods of remote sensing inversion of winter wheat seedling condition in the key period, and this research will provide timely support information and technology for farm production management. 【Method】Based on experimental data obtained from 2011-2013 in the fixed-point observation experiment, and using HJ-CCD satellite images, the quantitative correlations between key seedling condition parameters of winter wheat at booting stage in sampling regions and the grain quality parameters, production, and remote sensing variables were emphatically analyzed. In order to further enhance the mechanism and reproducibility of remote sensing inversion models, which were built and analyzed with ground measuring results, the quantitative level and reliability of remote sensing inversion models were raised. Models for monitoring the leaf area index, biomass, SPAD value, and leaf nitrogen content of winter wheat at booting stage using remote sensing variables extracted from the HJ-CCD images were built and assessed, respectively. 【Result】It is possible to invert leaf area index, SPAD value and leaf nitrogen content of winter wheat at booting stage by plant senescence reflectance index (PSRI), and invert biomass by ratio vegetation index (RVI), respectively. The remote sensing inversion models of the leaf area index, SPAD value, leaf nitrogen content and biomass of winter wheat were credible, and higher precision was obtained with determination coefficient (R²) of 0.651, 0.585, 0.630 and 0.675, respectively, and with root mean square error (RMSE) of 1.344, 4.62, 0.618% and 2 804.3 kg·hm^-2, respectively. It was especially reliable to inverse leaf nitrogen content by PSRI. According to the above results, the spatial distribution of the seedling condition parameters of winter wheat could be implemented with agricultural thematic maps of monitoring the key seedling condition parameters at different classes with remote sensing method, thus achieved quantitative expression of regional spatial distribution of the seedling condition parameters. It not only contributes to drawing up the plan of winter wheat field remedial measures and the water and nutrient resources scheduling, but also offers the decision basis for determination of agricultural policy and food trade. 【Conclusion】 The remote sensing inversion models of winter wheat key growth of seedling parameters at booting stage is feasible, and provide a quick, convenient and affordable method to extract the parameters seedling growth of large area for field production. The results of this research can provide timely valuable agricultural information for agronomists, agricultural departments, and farm managers.

The Relationship Between Lodging and Stem Endogenous Gibberellins Metabolism Pathway of Relay Intercropping Soybean at Seedling Stage

LUO Ling, YU Xiao-bo, WAN Yan, JIANG Tao, DU Jun-bo, ZOU Jun-lin, YANG Wen-yu, LIU Wei-guo

Scientia Agricultura Sinica. 2015, 48(13): 2528-2537. doi:10.3864/j.issn.0578-1752.2015.13.005

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【Objective】 From the perspective of endogenous gibberellins(GAs) metabolism, the reasons of soybean seedling vine lodging under the maize-soybean relay strip intercropping system were studied. 【Method】Under maize-soybean relay strip intercropping system and soybean sole cropping system, shade-tolerant and lodging resistant Nandou12 and non-shade tolerant and non- lodging resistant Nan032-4 were taken as experimental materials. The stalk morphology, anatomical structure, the level of gibberellin A₄, and the genes that are concerned with gibberellins expression profiles were analyzed with the paraffin section, Gas chromatography-mass spectrometry analyses, real-time qPCR with SYBR^®Green Ⅱ and other. methods. 【Result】Compared with soybean monoculture, due to the effects of maize shading at the seeding stage, both Nandou12 and Nan032-4 appeared obvious shade avoidance responses in relay strip intercropping system, including reduction of node number, significant increase of plant height and internode length, vining and lodging of the seedlings. However, the shade avoidance phenotypes were quite different between these two materials. Compared with the shade-tolerant cultivar Nandou12, the shade-sensitive Nan032-4 was influenced more seriously, for which 93.94% of the plants showed a lodging phenotype, much higher than that of Nandou12 (29.93%) (P＜0.05). The height of Nan032-4 was increased by 32.64 cm, different from that of Nandou12 (22.95 cm). The node number of stem reduced, but there was no significance between the two varieties. The upper nodes of Nan032-4 were longer than Nanou12 remarkably, that is to say, in the relay intercropping system, the increase of plant height of these two varieties resulted from internodes elongation, not from the increase of the node number, and the excessive elongation of middle and upper nodes could lead stems to vine and lodge easily. Vertical section of soybean stem revealed that the cell length of stems showed different degrees in elongation between the two varieties, especially for Nan032-4, not Nandou12, showed an apparent increase in cell length of stem pith, xylem and phloem. Therefore, these results suggest that the increase of plant height of these two varieties was caused by cell elongation, but not cell division. Endogenous gibberellins metabolism analyses showed the GA₄ content in the stems of both varieties was decreased significantly in relay strip intercropping system, but was much lower in Nandou12 compared to Nan032-4. The transcript levels of genes encoding key enzymes in gibberellins metabolic pathways were also investigated in the present study. The results show that the expression of genes encoding GA20-oxidase, GA3-oxidase, and GA2-oxidase were not only higher in Nan032-4 than that in Nadou12, but also up-regulated by intercropping in both varieties. However, GA synthetic genes of GmGA20ox5 and GmGA3ox6, as well as the GA deactivating gene GmGA2ox4 displayed a lower expression pattern in different plant modes and varieties. These results suggest that the content of endogenous gibberellins negatively regulate the expression of key genes in GA related pathways with a feedforward and feedback manner. Low level of GA₄ in the stem tips of Nandou12 might repress the stem elongation excessively, and contribute to a shade avoidance syndrome.【Conclusion】Synthesis of soybean endogenous GA₄ is affected by the genotypes and the light environment, thus influencing the growth and development of soybean stem, which finally results in the typical differences in shade lodging phenotype among varieties.

SSR Analysis of Population Genetic Structure of Rice Sheath Blight Causing Agent Rhizoctonia solani AG1-IA Collected from Eight Provinces (Autonomous Region) in Southern China

WANG Ling, ZUO Shi-min, ZHANG Ya-fang, CHEN Zong-xiang, HUANG Shi-wen, PAN Xue-biao

Scientia Agricultura Sinica. 2015, 48(13): 2538-2548. doi:10.3864/j.issn.0578-1752.2015.13.006

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【Objective】The objectives of this study are to investigate genetic structure of Rhizoctonia solani AG1-IA among different geographic populations from southern China, and to provide valuable information for the epidemiology of sheath blight disease on rice.【Method】R. solani AG1-IA population containing 188 isolates collected from 8 provinces (autonomous region) in southern China were genetically assessed using 8 fluorescence-labeled SSR markers. Genetic diversity parameters were calculated using POPGENE version 1.31, and inbreeding coefficient (F_IS) was generated with the computer program FSTAT 2.9.3. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by the Markov chain model with chi-square test implemented in the GENEPOP 4.2. Analysis of molecular variance (AMOVA) was carried out in the software Arlequin 3.1, and gene flow (Nm) was estimated according to population differentiation index (F_ST). To illustrate genetic relationships based on their pairwise Nei’s genetic distances, UPGMA dendrogram was constructed with MEGA 5.0. Bayesian clustering analysis was undertaken to determine the population structure and the degrees of genetic admixture using the STRUCTURE program version 2.3.3. Correlation between genetic and geographic distances between populations was assessed with a Mantel test. 【Result】 For populations of R. solani AG1-IA, the mean observed and effective number of alleles were 4.025 and 2.071, respectively. Shannon’s information index ranged from 0.659 to 1.088 with an average of 0.859. Allele richness varied from 2.500 to 5.152 with a mean of 3.858. Observed heterozygosity ranged from 0.425 to 0.619 with an average of 0.506, and the expected heterozygosity ranged from 0.399 to 0.546 with an average of 0.472. The negative value of inbreeding coefficient (F_IS=-0.069) indicated excess of heterozygotes (or deficiency of homozygotes) in the total population. Six of the eight populations significantly deviated from Hardy-Weinberg equilibrium due to heterozygote excess or deficiency, supporting the evidence for a mixed reproductive system in populations, including both asexual and sexual reproduction, while the balance between asexual and sexual reproduction was population dependent. AMOVA attributed about 88.14% of the variance to individuals within populations, indicating that the main genetic variation existed within populations. A positive correlation (r=0.422, P=0.025) was detected between genetic and geographical distances of populations by Mantel test. UPGMA dendrogram indicated that all populations were separated into two genetically differentiated subgroups (F_ST=0.209-0.624), the first included two populations (GN and CT) located along the Pearl River was significantly distinct from the second made up of six populations located along the Yangtze River, which in agreement with STRUCTURE-based analysis. Extensive genetic admixture was observed in the Yangtze River populations, and high levels of gene exchange occurring within the subgroup (Nm = 2.525-8.447) were indicated by low population differentiation (F_ST=0.029-0.094). 【Conclusion】 R. solani AG1-IA with a wide distribution, a mixed reproductive mode, and long-distance dispersal via sclerotia or mycelium, these characteristics could account for the relatively high level of genetic diversity among populations in southern China. The migration of R. solani AG1-IA, to some extent, to prevent genetic divergence of pathogen populations to ensure that high levels of gene flow occured in populations located in the Yangtze River tributary region. Significant population subdivision was evident between the Yangtze River populations and the Pearl River populations, suggesting that restricted long-distance migration of R. solani AG1-IA was a plausible explanation for the spatial structures of genetic variation of pathogen populations.

Analysis of the Genomic Location, Protein Structure Prediction and Expression of MAPK Gene StIME2 in Setosphaeria turcica

GONG Xiao-dong, WANG Yue, ZHANG Pan, FAN Yong-shan, GU Shou-qin, HAN Jian-min, DONG Jin-gao

Scientia Agricultura Sinica. 2015, 48(13): 2549-2558. doi:10.3864/j.issn.0578-1752.2015.13.007

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【Objective】The objectives of this study are to determine the location in genome, investigate the protein structure of StIME2 of Setosphaeria turcica, and to compare the expression level of StIME2 at different developmental stages and under different stress conditions, such as temperature, oxygen stress, hyperosmolar stress, hence laying a foundation for future studying functions of StIME2.【Method】 Blast search tool was used to determine the exact location of StIME2 in genome of S. turcica. Physical and chemical properties of StIme2 was analyzed by employing online software ProtParam, and secondary structure of StIme2 was predicted by applying Software SOMPA. Three-dimensional structure of StIme2 was doped out using PHYRE2 server and the structure model was evaluated using online software SAVES and the aim was to obtain Laplace image (Ramachandran Plot). StIME2 expression level was detected at different developmental stages and stress conditions based on semi-quantitative RT-PCR analysis. 【Result】The identity of StIME2 was 98 105, located between 1 560 184 and 1 562 574 in the positive-strand of scaffold_7 in the genome of S. turcica. StIme2 shared characteristic conserved domains of MAPK-like protein kinase, α-helices and random coils, which were its main elements of secondary structure of StIme2. β strands were less and mainly located in N-terminal in this structure. Three-dimensional structure of the protein was composed of a smaller N-terminal and a bigger C-terminal. The expression level of StIME2 was the highest in conidium developmental periods, and was the lowest during appressorium development stage. Under different stress conditions, the expression level of StIME2 appeared the highest when the incubation temperature was at 28℃. In hyperosmotic stress conditions, the expression level of StIME2 increased with the increase of NaCl concentration, but the expression level of the gene was almost completely inhibited at higher stress conditions (0.8 mol·L^-1 NaCl). Under oxygen stress conditions, in H₂O₂ stress treatment, the expression level of StIME2 was heightened with the increase of the concentration of H₂O₂. And the expression level of the gene was the highest under 10 mmol·L^-1 H₂O₂. 【Conclusion】 StIME2 was located between 1 560 184 and 1 562 574 in the positive-strand of scaffold_7 in the genome of S. turcica. The characteristics of StIme2 were analyzed, and the result showed that StIme2 shared all conserved domains of MAPK kinase of plant pathogenic fungi. However, unlike three types of MAPK genes, which were widely reported in plant pathogens, StIme2 was rarely reported. The expression level of StIME2 in conidium developmental period in S. turcica was the highest. Thus, the authors speculated that StIME2 could play an important role in regulating the developmental processes in spore of the pathogen. Moreover, the expression level of StIME2 changed with the temperature and the highest expression level was at 28℃. The gene may be also involved in osmotic stress and oxygen stress reaction of the pathogen.

Occurrence of Flixweed (Descurainia sophia) in Wheat Fields and Its Influence on Wheat Yield Components

FANG Feng, LI Mei, GAO Xing-xiang, LI Jian, LI Yan

Scientia Agricultura Sinica. 2015, 48(13): 2559-2568. doi:10.3864/j.issn.0578-1752.2015.13.008

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【Objective】Flixweed (Descurainia sophia) is the most serious broad-leaved weed in main areas of winter wheat production in China, and it seriously threatens winter wheat production. The objective of this study is to determine the D. sophia dynamics of emergence, plant height, fresh weight and its competitive effect on yield components of wheat. 【Method】 The dynamics of emergence, plant height and fresh weight of D. sophia were surveyed by fixed quadrat sampling and random sampling in wheat field with severe D. sophia infestation in Ji’nan from 2013 to 2014. Shallow rotary tillage was implemented in experimental plot with maize straw giving back before wheat sowing. In order to compare the effect of different D. sophia densities on wheat yield, three wheat planting densities were set as 67.5, 135.0, and 202.5 kg·hm^-2, each wheat planting densities set with different D. sophia densities as 0, 10, 20, 40, 60, 80, 160, 320, 640, and 1 280 plants/m². D. sophia densities were based on artificial inoculation, and three final thinning of seedlings were made before winter, early spring, and revival stage. Excel graphing was conducted to analyze the cause of yield loss caused by D. sophia.【Result】The peak of D. sophia seedling emergence appeared after wheat seeding one week to mid-November, the weekly average temperature at 13.5-14.8℃. The amount of seedling emergence before winter accounted for 96.7% of the total annual emergence. In late March, the weekly average temperature raised above 8.0℃, D. sophia seedling began to grow rapidly. Plant height of D. sophia was higher than wheat after early April. In mid-May, D. sophia plant height reached maximum 115.6 cm, and 43.4 cm higher than wheat. The fresh weight of D. sophia and wheat changed slowly at overwintering stage. Fresh weight of D. sophia increased rapidly after April, achieved the maximum 50.2 g in early May, which was four folds of wheat. The effect of D. sophia on wheat yield was primarily through inhibiting the effective ears and grain number per ear of wheat. It had no significant effect on 1 000-seed weight. Wheat spikes were from 4.29 to 0.28 million/hm², reduced by 93.5%, when the D. sophia plant rose from 0 to 640 plants/m² in the plot with 67.5 kg·hm^-2 wheat sowing amount. Wheat spikes were from 5.49 to 1.88 million/hm², reduced by 65.8%, when the D. sophia plant rose from 0 to 640 plants/m² in the plot with 135.0 kg·hm^-2 wheat sowing amount. Wheat spikes were from 6.69 to 3.22 million/hm², reduced by 52.0%, when the D. sophia plant rose from 0 to 320 plants/m² in the plot with 202.5 kg·hm^-2 wheat sowing amount. The loss rate of wheat was 84.7%, 71.9%, and 64.9% when the D. sophia density was at 320 plants/m² in the three wheat planting densities. The wheat yield was 2 396.3 and 1 680.2 kg·hm^-2, the loss rate was as high as 97.5% and 87.9% in the plots with wheat sowing amount at 67.5 and 135.0 kg·hm^-2, on the verge of failure. 【Conclusion】The dynamics of emergence, plant height and fresh weight of D. sophia were closely related with phenology. The damages of D. sophia can be effectively controlled and the impact of D. sophia on wheat production can be reduced by timely control and rational close planting.

Differences in Nitrogen Efficiency and Nitrogen Metabolism of Wheat Varieties Under Different Nitrogen Levels

WANG Xiao-chun, WANG Xiao-hang, XIONG Shu-ping, MA Xin-ming, DING Shi-jie, WU Ke-yuan, GUO Jian-biao

Scientia Agricultura Sinica. 2015, 48(13): 2569-2579. doi:10.3864/j.issn.0578-1752.2015.13.009

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【Objective】This study aims to clarify the difference in nitrogen metabolism of wheat varieties with different nitrogen use efficiencies and to achieve optimization of nitrogen application and high-yield.【Method】In this study, Luomai 18 and Yumai 49-198(LH), Xinong 509 and Yunong 202 (LL) were selected from 16 wheat varieties in yield conditions, and GS activity, soluble protein, free amino acids, NO₃^- and total nitrogen content of leaves were detected under nitrogen levels of N0 (0, CK), N120 (120 kg·hm^-2) and N225 (225 kg·hm^-2). 【Result】The results showed that GS activity, soluble protein, free amino acids, NO₃^- and total nitrogen content in leaf, N accumulation of aboveground and grain, yield and NPE of Luomai 18 and Yumai 49-198 (LH) were significantly higher than that of Xinong 509 and Yunong 202 (LL). GS activity, soluble protein, free amino acids, NO₃^- and total nitrogen content in leaf, N accumulation of above ground and grain, yield increased with the increasing of nitrogen level, and the NPE reduced. But the response of the two types of varieties to nitrogen levels was different, compared with N0, the growing rate of GS activity, soluble protein, free amino acids, NO₃^- and total nitrogen content in leaf, N accumulation of above-ground of Xinong 509 and Yunong 202 (LL) were significantly higher than that of Luomai 18 and Yumai 49-198(LH), however, the growing rate of yield of Xinong 509 and Yunong 202 (LL) were significantly lower than that of Luomai 18 and Yumai 49-198(LH); the decreasing range of NPE of Luomai 18 and Yumai 49-198 (LH) was significantly higher than that of Xinong 509 and Yunong 202 (LL). 【Conclusion】Compared with Xinong 509 and Yunong 202 (LL), the reason that Luomai 18 and Yumai49-198 (LH) had higher yields and nitrogen use efficiency was that they had higher GS activities. Therefore the ability of nitrogen uptake and assimilation were improved, the nitrogen physiological efficiency in the whole nitrogen metabolism process was increased and better yields were obtained. Increasing the supply of nitrogen, yields of Luomai 18 and Yumai49-198 (LH) were significantly promoted. Luomai 18 and Yumai49-198 (LH) have a stronger tolerance to low nitrogen stress and the potential of yield is greater; Xinong 509 and Yunong 202 (LL) are more sensitive to nitrogen, but their ability in distribution of N is lower.

Effect and Mechanism of Maize Intercropping with Soybean on Reducing Soil Nitrogen Residue in Wheat-Maize Rotation

ZHANG Yi-tao, REN Tian-zhi, LIU Hong-bin, LEI Qiu-liang, ZHAI Li-mei, WANG Hong-yuan, LIU Shen, YIN Chang-bin, ZHANG Ji-zong

Scientia Agricultura Sinica. 2015, 48(13): 2580-2590. doi:10.3864/j.issn.0578-1752.2015.13.010

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【Objective】 The purpose of this study was to clarify the impact of maize intercropping with soybean on the yield, nitrogen absorption content, soil water content and nitrate residue in wheat-maize rotation system. And the relative contribution of above-ground and below-ground to intercropping advantage was also distinguished, to provide a reference for improving soil productivity and optimizing resource allocation. 【Method】 Four treatments were set up in representative farmlands in Xushui County of Hebei Province from June 2011 to October 2012, including sole maize (T1), sole soybean (T2), maize intercropping with soybean but no root separation (T3), and maize intercropping with soybean with root separation (T4). Crop growth, soil water content and nitrate N concentration in several critical growth periods were monitored in real-time. 【Result】 Intercropping had obvious advantage compared to monoculture. Land equivalent ratio (LER) of maize intercropping with soybean was greater than 1, and its total N accumulation (256.1 kg·hm^-2) was significantly higher than sole maize (159.7 kg·hm^-2). Maize intercropping with soybean improved the system capacity mainly by promoting maize growing and nitrogen accumulation, of which above-ground could increase intercropped maize of biomass, yields and N accumulation by 81.6%, 83.4% and 75.7%, respectively, while the contribution of below-ground was only 18.4%, 16.6% and 24.3%, respectively. Soil water content of intercropped maize was significantly lower than that of sole maize, and soil water content of intercropped maize with root separation was significantly lower than that of intercropped maize without root separation. There was no significant difference in soil water content between intercropped soybean and sole soybean, while root separation had no significant effect on soybean. Compared to monoculture, intercropping system reduced soil nitrate N content of intercropped maize while increased that of intercropped soybean. Compared to no root separation, root separation had a slight effect on soil nitrate N content of intercropped maize but significantly decreased soil nitrate N content of intercropped soybean. No matter monoculture or intercropping in summer, yield and nitrogen accumulation by crop of their residual crop (wheat) had no significant difference, but intercropping could significantly reduce soil nitrate residue after wheat harvest (P＜0.05). Soil (0-100 cm) nitrate N residue of intercropping after wheat harvest reduced by 87.2 kg·hm^-2compared to sole maize, but the contribution of above-ground was 77.5% and that of below-ground was only 22.5%. 【Conclusion】 Intercropping in summer had significant yield advantages, and its N accumulation was higher than sole maize, of which the contribution of above-ground was greater than below-ground in intercropping system, while there was no significant impact on the succession wheat yield and N accumulation. Compared to monoculture, intercropping reduced soil water content of intercropped maize but had no effect on intercropped soybean, and soil nitrate N content of intercropped maize significantly reduced but that of intercropped soybean significantly increased, however, intercropping system could reduce soil nitrate residue of both the current and after-crop harvest.

Relationship Between Fruit Softening and the Metabolism of Sugar and Starch and Their Related-Gene Expression in Post-Harvest ‘Jingbaili’ Fruits

QI Xiu-dong, WEI Jian-mei, ZHAO Mei-wei, PENG Hong-li, ZHANG Hai-e

Scientia Agricultura Sinica. 2015, 48(13): 2591-2599. doi:10.3864/j.issn.0578-1752.2015.13.011

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【Objective】The aim of this study is to investigate the relationship between sugar and starch metabolism and fruit softening so as to provide a basis for developing better storage technology in ‘Jingbaili’ fruits. 【Method】‘Jingbaili’ pear fruits, treated at low temperature and with 1-MCP, were taken as the sample materials to determine the firmness, respiration rate, sugar and starch contents and the activities of related enzymes, and the key enzymes gene expression (AM, SPS, SS, and AI) were analyzed by real-time fluorescence quantitative PCR during fruit ripening and softening. 【Result】During storage of ‘Jingbaili’ fruits, the starch contents decreased rapidly, showing a positive correlation significantly with firmness, and this trend was dramatically inhibited at low temperature with 1-MCP treatment. Also, the activity of amylase (AM) increased rapidly, which was significantly related with the changes of firmness and starch contents, and AM gene expression also accumulated rapidly with fruit softening. These processes showed a strongest effect at low temperature with 1-MCP treatment. During storage, only the glucose contents showed a significant decreasing trend, the contents of fructose and sucrose increased slightly, but they were inhibited at low temperature with 1-MCP treatment. Among sucrose-metabolized enzymes, only acid invertase (AI) activity was significantly correlated with firmness, but its activity and gene expression increased mainly at late stage of storage, lagging behind AM. Sucrose phosphate synthase (SPS) and sucrose synthase (SS) activities were not correlated significantly with firmness, but they showed higher activity and gene expression, and had a close relationship with the contents of sucrose and fructose, significantly regulated at low temperature with 1-MCP treatment. 【Conclusion】There was a strongest relationship between starch degradation and ‘Jingbaili’ fruit softening, AM was an important enzyme involving at early softening stage. Sucrose metabolism involved in fruit ripening and softening, but AI mainly affected the softening process at late stage and both SPS and SS were perhaps involved in the physiological processes of softening by means of regulating the composition and contents of the soluble sugars during fruit softening.

Analysis of Anthocyanins Related Compounds and Their Biosynthesis Pathways in Rosa rugosa ‘Zi zhi’ at Blooming Stages

ZHANG Ling, XU Zong-da, TANG Teng-fei, ZHANG Hui, ZHAO Lan-yong

Scientia Agricultura Sinica. 2015, 48(13): 2600-2611. doi:10.3864/j.issn.0578-1752.2015.13.012

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【Objective】The composition content and structure of pigments in Rosa rugosa ‘Zi zhi’ and their biosynthetic pathways were studied in order to lay a foundation for further investigation of the floral pigment mechanism and molecular breeding of R. rugosa. 【Method】 Anthocyanins, flavonoids and carotenes in the petals of flowers at different blooming stages were determined using HPLC and ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-DAD-Q-TOF-MS). The biosynthetic pathways of anthocyanins in R. rugosa ‘Zi zhi’ were proposed according to the results of HPLC, UPLC-DAD-Q- TOF-MS and chemical reactions. 【Result】 In all, 8 kinds of anthocyanins, 16 kinds of flavones and flavonol glycosides and β-carotene were detected from the petals of R. rugosa ‘Zi zhi’, but xanthin was not detected. The main structures of the anthocyanins were bioside of peonidin, delphinidin, cyanindin and pelargondin. The highest relative contents of the four anthocyanins were detected at budding-flower and initial flowering stages. Two types of methylation of paeonidin were detected while no methylation was detected in delphindin. The main compounds of flavonoid were glycosylations, acylations, methylated quercetin and kaempferol. The results of quantitative analysis showed that the contents of paeonidin and delphindin accounted for more than 90% of the total anthocyanins. The content of paeonidin was reduced with the color fading while the content of delphindin was stable at all stages and the ratio of paeonidin to delphindin was reduced with the flower blooming.【Conclusion】There are peonidin, delphinidin, cyanindin and pelargondin in the petals of R. rugosa‘Zi Zhi’. The four anthocyanins are synthesized at the budding-flower and initial flowering stages, and the contents of them are reduced during the flower blooming. Peonidin is the main composition that determines the flower color of R. rugosa‘Zi zhi’. The catalytic reactions of RrAOMT exhibit substrate specific patterns.

Drying Characteristics and Model of Walnut in Air-Impingement Jet Dryer

ZHAO Ke, XIAO Xu-lin

Scientia Agricultura Sinica. 2015, 48(13): 2612-2621. doi:10.3864/j.issn.0578-1752.2015.13.013

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【Objective】 In order to improve the dried walnut quality, shorten the drying time, the effects of different conditions on the walnut air-impingement jet drying were studied and the activation energy for drying was obtained and the optimum drying model was selected.【Method】 Using the method of energy saving technology of dry heat pipe combined with a self-made gas jet impingement equipment, in 9 groups of an experiment, effects of different jet air temperatures (40℃, 50℃ and 60℃) and air velocities (11, 12 and 13 m·s^-1) on the drying characteristics of materials, effective moisture diffusion coefficient and activation energy were studied, at the same time, through the statistical data selection for the fitting drying models, 5 drying kinetics models were established. The 5 models are the Page model, the Modified Page model, the Logarithmic model, the Herdenson and Pabis model, and the Lemus model. The data were processed by using the DPS, after finishing the fitting, a parametric equation was obtained the final general MR and t. 【Result】 Compared to the most results of food material drying test, air-impingement jet drying of walnut mainly occurred in the falling rate drying period, and there was no constant drying rate stage. Air temperature had large influence on each stage of walnut air impingement. With the increase of the air temperature values, the drying rate was rising and the MR was decreased. The air velocity almost had no influence on the drying time, but had a certain influence on the rate of surface water vaporizing stage by increasing the drying time. The air velocity almost had no influence on the drying rate of internal moisture transfer stage had almost no influence, and by employing this feature, a method of changing the wind temperature and wind speed could be used in different periods, so not only the drying time was shortened, but also the purpose of energy saving was achieved. Overall, for shortening the drying time, the order of the influence was air temperature＞wind speed. The drying rate of the air-impingement jet drying of walnut increased with the increase of air temperature, while had no effect by the air velocity increasing. Fick’s second law was used to calculate the effective moisture diffusivity of walnut in drying process and the values were in the range of 0.9674×10^-11 to 2.2231×10^-11m²·s^-1. Because of its shell structure so that the effective moisture diffusion coefficient was 1-3 orders of magnitude lower than other food materials. The activation energy was increased with the increase of the air velocity and the lowest activation energy was 27.644 kJ·mol^-1. The five models had a higher fitting degree, and could better describe the walnut air impingement drying. The Modified Page model gave the highest coefficient of determination (R²), the lowest chi-square (χ²) and root mean square (RMSE). Based on the Modified Page model, regression analysis by using the DPS, a parameter equation was established between generally applicable moisture ratio (MR) for walnut air impingement drying and time (t) in the circumstance where the temperature of wind is 40-60℃ and the speed of wind is 11-13 m·s^-1. 【Conclusion】 Air temperature and air velocity had influence on the drying curve, the drying rate curve, effective moisture diffusion coefficient and activation energy. According to the fitting result of the fitted values and the observated values under different conditions, the optimal drying condition was that the air temperature is 50℃ and the wind speed was 13 m·s^-1. The Modified Page and Page models could properly describe the air-impingement jet drying behavior of walnut and could be used when the drying air temperature between 40 to 60℃, air velocities between 11 to 13 m·s^-1. The Modified Page model fitting degree higher, it is the optimal model of the walnut air impingement drying.

A Bibliometric Analysis on Technology Innovation of Livestock Breeding

WEI Xun, JIA Jing-dun, SUN Kang-tai, GE Yi-qiang

Scientia Agricultura Sinica. 2015, 48(13): 2622-2634. doi:10.3864/j.issn.0578-1752.2015.13.014

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【Objective】In order to summarize the research history, understand the frontiers and focus and provide a sound foundation for researchers and policy-makers, the comparative analysis of world literature and patents about technology innovation of livestock was carried out. 【Method】Based on Science Citation Index and Derwent Innovation Index database, with bibliometric methods, relevant literature and patents on livestock breeding during the years of 1964 and 2014 were collected. Thomson Data Analyzer, Thomson Innovation, OSviewer and other tools were used. 【Result】Studies on livestock breeding technology have gone through three stages: the start-up and slowly developing phase lasted from 1960 to 1990, focusing on performance determination evaluation; the rapid growth phase lasted from 1990 to 2000, with the wide use of BLUP breeding, artificial insemination, superovulation and embryo transfer technology，the leaping forward phase began in 2000 till now, gender control technology; molecular markers and genomic selection technology boost the development of research, but the patent application experienced fluctuations in the past decade. The United States possesses the prominent capability in the field all over the world, reflected by the amount of paper issued, citations, and European developed countries and Japan have long occupied a leading position while research from China go through a rapid development in recent years, particularly in terms of patents, but the quality of Chinese research needs to be improved according to the average citation. Ten out of top 20 patent applicators are from US, all of which are enterprises while China has 4 universities and institutions in the top 20, indicating that innovation system with the enterprises as the mainstay has not been established in China. Cattle and pigs have been the focus in the field. Research on cattle developed in 1990’s, but was catch up by that on pig after 2000; and research on sheep has a rapid and consistent growth while that the development on horse was slow; finally, research on donkey and camel was always relatively rare. Research about propagation techniques started earlier, with the largest number of literature, but the trend showed greater volatility even in shock down in recent years after the heyday of sex control technology during 1990’s; research on genomics techniques increased rapidly after the completion of gene draft of certain species in 2000, promoting the development of proteome, transcriptome, methylation group and metabolomics group; genetic manipulation techniques transferred from the study of single gene transfer, gene knockout and knock-in, gene targeting technology to bioreactor technology since 1991, and transferred further to TALEN technology, genome editing technology after 2004. Genetic marker technology, as the key of precision breeding, developed from protein level to DNA molecular level since 1991, grew rapidly after 2004 due to the increasing of maker types and the application of SNP marker, and after 2008, the precision breeding technology became the most focused area instead of propagation techniques.【Conclusion】Pigs and cattle are still the main objects of study, precision breeding technique now has become the main method of current research represented by whole genome selection, and nucleic acid assay or test methods are the main directions of patent applications. The United States in this field has outstanding leadership, while Germany, Britain, France, Japan and other developed countries also have a good research base and patent applications situation. The research in China is developing rapidly in recent years, and even has the opportunity of catching up; however, the quality of research still needs to be strengthened.

Effects of Illumination Time and Ambient Temperature on Reproductive System and Gene Expression and Secretion of Hormone in Breeding Geese

YANG Hai-ming, JU Xiao-jun, WANG Zhi-yue, DING Jia-tong, WANG Xin-xi, CHEN Yong-hua

Scientia Agricultura Sinica. 2015, 48(13): 2635-2644. doi:10.3864/j.issn.0578-1752.2015.13.015

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【Objective】The experiment was conducted to study the effects of illumination time and ambient temperature treatments on reproductive system and gene expression and secretion of hormone in breeding Yangzhou geese, so that the functional characteristics of illumination time and ambient temperature could be preliminarily discovered. 【Method】 The geese of 200-day-old and receiving gradually decreased natural sunlight were selected as experimental animals. At the beginning of experiment, the sunlight time was 9.7 h a day, and the ambient temperature was about 5℃. Treatments were assigned in a completely randomized design with factorial arrangement (2 × 3) including 2 levels of ambient temperature (0-5℃, 25-30℃) and 3 levels of illumination time (8 h, 12 h, 16 h). The illumination increased and decreased based on the natural day. In this study 120 adult female geese were randomly assigned to 6 groups with 20 birds each. Feed and water were freely available. On day 30, 5 geese of each treatment were taken randomly to collect 3 mL blood sample from wing venous for determination of related hormone indexes. At the same time, 3 geese of each treatment were got randomly to be slaughtered and hypothalamus and pituitary were collected quick-freezed and then conserved in ultra-low-temperature storage for determination of related gene expression. The reproductive system was separated to determine weight or length, simultaneously oviduct magnums were taken for tissue slice. 【Result】Serum concentrations of luteinizing hormone (LH) in 16 h illumination treatments were significantly more than those in 8 h and 12 h illumination treatments (P＜0.05). Serum concentrations of prolactin (PRL) in 16 h illumination treatments were significantly more than those in 12 h illumination treatments (P＜0.05). Serum concentrations of PRL in high temperature treatments (25-30℃) were significantly higher than those in low temperature treatments (0-5℃) (P＜0.05). There were significant interactions on serum concentrations of LH, PRL and estradiol (E₂) between illumination time and ambient temperature (P＜0.05). The ovary index and follicle index of 12 h illumination treatments were significantly more than those of 16 h illumination treatments (P＜0.05). The follicle number of 8 h illumination treatments was significantly more than that of 16 h illumination treatments (P＜0.05). The oviduct length of 12 h illumination treatments was significantly more than those of 8 h and 16 h illumination treatments (P＜0.05), and the oviduct index of high temperature treatments was significantly higher than that of low temperature treatments (P＜0.05). There were no significant effects on ovary index, follicle index, follicle number, oviduct index and oviduct length under different ambient temperatures (P＞0.05). The oviduct magnum of 8 h and 12 h illumination treatments and the high temperature treatments showed scarlet. In addition, the pseudostratified columnar epithelium and lamina propria were thicker, morever their rugas were obvious, and the secretory glands were rich. However, the oviduct magnum of 16 h illumination treatments and the low temperature treatments showed dull red. Their pseudostratified columnar epithelium and lamina propria were obviously thinner, furthermore rugas were fuzzy, and the secretory glands were less. Relative expression quantity of PRL gene of 16 h illumination treatments was significantly higher than that of 8 h and 12 h illumination treatments (P＜0.05). There were no interactions on reproductive system, mRNA expression and secretion of hormone in breeding geese (P＞0.05). 【Conclusion】Long time illumination of 16 h suppressed growth of ovarian and follicle, and illumination of 12 h increased oviduct length. Long time illumination of 16 h increased expression abundance of PRL gene. Therefore, long illumination time depressed reproductive function, and the optimum illumination time of geese was 12 h. High temperature increased serum concentration of LH and PRL, and the organization structure of breeding geese were affected by low temperature, so the ambient temperature should be controlled, and the breeding geese need to avoid being raised in the extreme temperature environment.

Identification and Analysis of the Characteristics of Spatial and Temporal Expression of Bombyx mori Cysteine Protease Inhibitor BmCPI40

LI Jian-wei, LI Yi, ZHOU Xiao-ying, LI Zhi-lang, CHEN Shi-da, TIAN Sha, HOU Yong, XIA Qing-you

Scientia Agricultura Sinica. 2015, 48(13): 2645-2655. doi:10.3864/j.issn.0578-1752.2015.13.016

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【Objective】 The objective of this study is to identify a newly discovered cysteine protease inhibitor of silkworm (Bombyx mori) and to investigate the expression characteristic and regulation mechanism, thereby providing data for further investigation on the inhibitor functions in the development process of molting and metamorphosis in the silkworm. 【Method】 Primers were designed using Primer Premier 5.0 software according to the Silkworm Genome Database, and were used to amplify the sequence of BmCPI40 that the putative signal peptide was eliminated. The multiple sequence alignment and phylogenetic analysis of BmCPI40 was performed by bioinformatics software Clustal X and MEGA 4.0. The recombinant BmCPI40 protein was expressed in prokaryotic expression system, and then was used to produce the polyclonal antibody after purification. The spatial and temporal expression pattern of BmCPI40 was investigated using RT-PCR and quantitative real-time PCR (qPCR), and the protein distribution and localization in different tissues and different developmental stages was analyzed using Western blot and immunofluorescence. In addition, the 20E-induced expression of BmCPI40 was also studied after injection using qPCR. Furthermore, the promoter activity of BmCPI40 was analyzed in silkworm cells with Dual Glo Luciferase Assay System. 【Result】A new cysteine protease inhibitor of silkworm, named BmCPI40, was cloned and expressed. The open reading frame of BmCPI40 was 366 bp that encoded a protein with 121 amino acid residues, the first 18 residues of which was predicted as signal peptides, and the isoelectric point was 4.43. Phylogenetic analysis indicated that this inhibitor showed high sequences identity with the propeptides of several microbes cysteine proteinases. Soluble protein was produced from recombinant E. coli cell. The spatial expression results suggested that BmCPI40 was expressed high in the epidermis and weakly in the head. The temporal expression indicated that the expression of BmCPI40 in the larvae stage was significantly higher than that in the pupal stage, while the expression was significantly reduced during molting and metamorphosis. Immunofluorescence experiments showed that BmCPI40 was synthesized in the epithelial cells. Moreover, it was found that BmCPI40 could be inhibited by ecdysone, because that its expression level decreased after injection with 20E in the silkworm and promoter activity also decreased after induction with 20E in the BmE cell. 【Conclusion】Ecdysone induced the decrease of expression level of BmCPI40, which may be involved in the molting and metamorphosis in the silkworm.

Comparison Analysis of Starch and Protein Expression Profiles on Cassava Tuberous Roots cv. SC8 and Its Tetraploid

AN Fei-fei, CHEN Song-bi, LI Geng-hu, ZHOU Kai, LI Kai-mian

Scientia Agricultura Sinica. 2015, 48(13): 2656-2665. doi:10.3864/j.issn.0578-1752.2015.13.017

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【Objective】The aim of the study was to better understand the change of starch content, starch structure and its proteins regulatory mechanisms of cassava tuberous roots of diploid and its tetraploid.【Method】Tuberous roots of 10 months after planting were used as the materials, starch content was measured by weight in air and water, the proportion of amylose and amylopectin was determined by spectrophotometry, Excel 2013 and DPS v7.05 statistical software were used to analyze the data, the significant difference was determined by using Duncan method, the size, shape and number of amyloplast were observed by scanning electron microscopy, Western blot was used to verify some enzymes involved in starch synthesis and degradation, roots proteins of cassava tetraploid and diploid genotypes were excised and purified from 2D gels, Delta 2D software were used to analyze the different proteins with more than 2.0 fold changes and assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF-MS/MS) were used to identify them, and the proteins were classified according to their functions combined with KEGG database.【Result】After chromosome doubling in tetraploid cassava, dry matter content, starch content and fresh roots weight were significantly decreased by 12.18%, 11.41%, and 35.34%, respectively. No significant difference was observed in amylase and amylopectin proportion. No significant difference in amyloplast morphology, mainly spherical, irregular sphere and ellipsoid also exist, the size was uniform, while the loose degree of amyloplast and space gap were increased, the number was decreased. The level of sucrose phosphate synthase (SPS) significantly decreased, β-amylase expression was increased, while the level of granule bound starch synthase I (GBSSI) was not changed. Twenty differential expressed protein spots were showed and analyzed by software, including 2 up-regulated spots and 18 down-regulated spots, 19 were successfully matched in database, while spot 16, which was down-regulated, was no match. The functions of the 17 down regulated proteins were associated with carbohydrate and energy metabolism (4), structure (3), DNA and RNA metabolism (2), chaperones (2), HCN metabolism (2), antioxidant (1), protein synthesis (1) and unknown function (2), 2 up-regulated proteins were stem-specific proteins (TSJT1).【Conclusion】Protein related with carbohydrate and energy metabolism, nucleic acid metabolism and chaperones were down-regulated, the amyloplast loosely arranged and number decreased, the expression level of SPS decreased, β-amylase expression increased, indicating the low starch synthesis ability and high starch decomposition ability in tetraploid cassava, then decreased starch content, while no significant changes in amylose and amylopection proportion was found.

Effects of Nitrogen Fertilizer of Different Forms and Ratios on the Growth, Nitrogen Absorption and Utilization of Young Navel Orange Trees Grafted on Poncirus trifoliata

FAN Wei-guo, GE Hui-min

Scientia Agricultura Sinica. 2015, 48(13): 2666-2675. doi:10.3864/j.issn.0578-1752.2015.13.018

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【Objective】Cultivation media have important influence on the citrus absorbing different forms of nitrogen. Calcareous yellow soil is the main soil type in Karst area, southwest of China, and citrus are widely distributed in this region. Research on the citrus absorption and utilization of different forms of nitrogen will provide a reasonable scientific method of selecting nitrogen fertilizer for citrus in calcareous yellow soil in this region. 【Method】 A soil culture experiment was conducted to determine the total leaf area, plant height, base diameter, fresh weight, dry biomass, root/cap ratio, nitrogen absorption and utilization efficiency of young Newhall navel orange with different N treatments: single NO₃^--N, single NH₄⁺-N and different proportions mixture of both forms, which were grafted on Poncirus trifoliata and cultivated in the calcareous yellow soil. The conventional method of depletion was carried out to study the kinetics parameters of NO₃^--N and NH₄⁺-N absorption in both spring and summer.【Result】Results indicated that plant growth was obviously inhibited, and the total leaf area, height, base diameter, fresh weight and dry weight biomass, root-shoot ratio, nitrogen absorption amount and nitrogen use efficiency were brought down when using NO₃^--N, NH₄⁺-N and CO(NH₂)₂ alone. All these indexes were the lowest when applying CO(NH₂)₂ alone. Under mixed supply of sodium nitrate and NH₄⁺-N, the plant growth, nitrogen absorption amount and nitrogen use efficiency were better than using them alone. When the proportion of sodium nitrate﹕NH4⁺-N reached 75﹕25, growth indexes, nitrogen absorption amount and nitrogen use efficiency were the highest, the total leaf area was 0.44 m², height was 73.95 cm, base diameter was 1.36 cm, fresh weight was 232.95 g/plant, dry weight biomass was 130.27 g/plant, the ratio of fresh weight and root-shoot was 1.02 and the ratio of dry weight and root-shoot was 1.06, nitrogen absorption amount and nitrogen use efficiency were 3.80 g/plant and 0.0292 g·mg^-1, respectively. Under mixed supply of sodium nitrate and NH₄⁺-N, the plant growth, nitrogen absorption amount and nitrogen use efficiency were decreased when the ratio of sodium nitrate increased, the ammonia poisoning phenomena happened intheroot system of navel orange young trees when using NH₄⁺-N or CO(NH₂)₂ alone. No matter in spring or summer, there was no obvious difference in maximum absorption rate (I_max) between fertilizing NO₃^--N alone and mixed different ratios of NO₃^--N and NH₄⁺-N, however, the absorption of NO₃^- was more stable than the NH₄⁺. The I_maxof NO₃^- in spring was smaller than in summer, but the affinity of root to NO₃^- was stronger than in summer, and the K_m of root to NO₃^- was no significant difference in summer. When the ratio of NO₃-N and NH₄⁺-N was up to 50%-75%, the affinity of root to NO₃^- could be enhanced in spring, and the flow rate of NO₃^-(α) could also be increased in both spring and summer. In spring, the maximum absorption rate and the flow rate of root to NH₄⁺ were both increased with the increasing ratio of NH₄⁺-N, but the affinity of the root to NH₄⁺ decreased. In summer, the maximum absorption rate, affinity and the flow rate of root to NH₄⁺ were all reduced with the raising of the ratio of NH₄⁺-N, and the lowest values of them were observed when using NH₄⁺-N alone. Mixed fertilizer of NO₃^--N and NH₄⁺-N, the affinity of root to NO₃^- was stronger than to NH₄⁺, as well as the α of NO₃^-was faster than NH₄⁺ in both spring and summer. 【Conclusion】The root system of young navel orange trees grafted on Poncirus trifoliata prefered absorption NO₃^-in calcareous yellow soil. The proportion of sodium nitrate and NH₄⁺-N application reached 75﹕25 was able to promote the plant growth and nitrogen absorption and increase the use efficiency for navel orange.

Study on the Interspecies Crossbreeding Between Gayal (Bos frontalis) and Yellow Cattle(Bos taurus)

HE Zhan-xing, WANG Peng-wu, LUO Zai-ren, LI Chun-mei, YIN Yi-chang, QU Kai-xing, WANG An-kui, ZHANG Ji-cai, HUANG Bi-zhi, YUAN Xi-ping

Scientia Agricultura Sinica. 2015, 48(13): 2676-2686. doi:10.3864/j.issn.0578-1752.2015.13.019

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【Objective】To investigate the utilization and prospects in crossbreeding between the Gayal (Bos frontalis) and Yellow cattle (Bos taurus). 【Method】Interspecies crossbreeding were carried out on Yunnan yellow cattle(YY, n=379) and Simmental crossbred cattle (Simmental ♂× Yunnan yellow cattle ♀, SY, n=136) as the female parent inseminated artificially with frozen semen of Gayal. Pregnancy test of cow at about 60 days, number of calving were tracked, and body weight, body measurement, appearance characteristics of crossbred F₁ were investigated. 【Result】Results were showed that: ① Rates of average conception and calving from 515 cows with frozen sperm of Gayal were 73.98% and 92.13%，thereinto rates of average conception and calving were 68.87% and 90.81% in YY, and 88.24% and 95% in SY, respectively. ②The body weights of crossbred F₁ (Gayal × SY, MSY) at birth, 6-month, 12-month, 18-month and 24-month of age were 22.86, 103.86, 142.4, 176.21 and 243.25 kg, respectively, which were 94.89%, 26.01%, 31.04%, 41.42%, and 84.14% significantly higher than those of YY(P＜0.01), and 29.15%(P＜0.01), 1.63%(P＞005), 5.63%(P＞0.05), 9.16%(P＜0.05) and 20.47%(P＜0.01) higher than those of MY; Body weights of crossbred F₁(Gayal × Yunnan yellow cattle, MY) at birth, 6-month, 12-month, 18-month and 24-month of age were in turn 17.7, 102.19, 134.81, 161.43 and 201.91 kg, which were 50.90%, 23.99%, 24.40%, 36.83% and 52.85% significantly higher than those of YY(P＜0.01 or 0.05); Average daily gain(ADG) of MSY from birth to 24-month age was 301.90 g, which was significantly higher than that of MY (264.67 g) and YY (164.89 g) (P＜0.01), and ADG of MY was also significantly higher than that of YY(P＜0.01); heterosis rates of birth, 12-month and 24-month age of MY were 28.21%, 3.87% and 4.20%, respectively, especially heterosis of birth stage was the highest, which was more distinct than that of other age stage. ③Withers height and heart girth at birth were 59.09 and 60.09 cm in MSY and 60.78 and 59.15 cm in MY, which were significantly higher than those of YY(P＜0.05); At 6 months of age, withers height, heart girth, abdomen girth and circumference of cannon bone of MSY were 106.47, 118.67, 125.18 and 13.66 cm, respectively, which were 25.88%, 16.50%, 9.81% and 29.48% higher than those of YY(P＜0.05), and heart girth, abdomen girth and circumference of cannon bone of MY were 112.95, 126.65 and 12.79 cm, respectively, which were 10.89%, 11.10% and 21.23% higher than those of YY(P＜0.05); At 12 months of age, body length, withers height, heart girth, abdomen girth and circumference of cannon bone of MSY were in turn 124.04, 125.00, 148.00, 165.08 and 14.60 cm, which were 33.82%(P＜0.05), 30.64%(P＜0.05), 31.16%(P＜0.01), 29.95%(P＜0.01) and 26.63%(P＜0.05) higher than those of YY, and heart girth, abdomen girth and circumference of cannon bone of MY were 125.72, 143.98 and 14.05 cm, which were 11.41%(P＜0.05), 13.34%(P＜0.01) and 21.86%(P＜0.05) higher than those of YY; body length, withers height, heart girth and abdomen girth of MSY were increase at 33.20%(P＜0.05), 31.43%(P＜0.05), 17.72%(P＜0.01) and 14.65%(P＜0.01) higher than those of MY; At 24 months of age, body length, height at withers, heart girth, abdomen girth and circumference of cannon bone of MSY were 147.00, 134.75, 163.50, 179.50 and 16.85 cm, respectively, which were 33.30% (P＜0.01), 31.69% (P＜0.01), 38.83%(P＜0.01), 32.12% (P＜0.01) and 41.60% (P＜0.05) higher than those of YY; Heart girth, abdomen girth and circumference of cannon bone of MY were 144.49, 169.04 and 16.77, which were 22.69%(P＜0.01), 24.42% (P＜0.01) and 40.92% (P＜0.05) higher than those of YY; Body length, height at withers and heart girth of MSY were increased at 38.72%, 27.83% and 13.16% higher than those of MY (P＜0.01). It indicated crossbred F₁ showed distinctly high on withers height and heart girth at birth, and distinctly advantages on heart grith, abdomen girth and circumference of cannon bone after 6 months of age, especially body length, withers height and heart girth of three-way crossing were more obvious growth than those of two-way crossing from the 12 months of age. ④Birth weight of crossbred F₁ from Gayal and Dali yellow cattle was significantly higher than that of crossbred F₁ from Gayal and Dehong yellow cattle, and various body measurements were not significantly difference between two crossbred F₁. It indicated the effect of crossbreed was basically the same between Gayal × Dali yellow cattle and Gayal×Dehong yellow cattle. ⑤The mainly habit of adult crossbred F₁ was highly the similar as Gayal. 【Conclusion】The results showed that interspecies hybridization of Gayal and Yunnan yellow cattle is viable, which put up good character such as fast growth, strong adaptability, distinct heterosis. Especially, heterosis of three-way crossing was better than that of two-way crossing; Crossbred F₁ cow was fertile ability, but the crossbred F₁ male was maybe infertile. Finally, interspecies hybridization of Gayal and Yunnan yellow cattle was good at utilization potentiality, popularization and application.

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