Scientia Agricultura Sinica

Analysis of Maize Grain Filling Rate in Different Heterotic Groups

ZHANG Dong-mei, LIU Yang, ZHAO Yong-feng, ZHU Li-ying, HUANG Ya-qun, GUO Jin-jie, CHEN Jing-tang

Scientia Agricultura Sinica. 2014, 47(17): 3323-3335. doi:10.3864/j.issn.0578-1752.2014.17.001

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【Objective】The objective of this experiment was to study the characteristics of grain filling rate of different heterotic groups and to select the excellent inbred lines with fast grain filling rate, providing the reference for the selection of high yield maize hybrids.【Method】A total of 173 maize inbred lines were used as experimental materials. The drying method was used to determine grain filling rate of 10, 20, 30, and 40 days after pollination and 6 related correlation characters. The combined analysis of variance for year, lines, sample time, repetition, year×lines, lines×sample time, year×lines×sample time were carried out by SAS software; and the SPSS software was used to perform the correlation analysis on the grain filling rate and its related traits such as the husk moisture content 10, 20, 30, and 40 days after pollination, the number of husks, the cob moisture content 40 days after pollination, the cob length, the cob diameter and the grain moisture content 40 days after pollination. A genome-wide scan was performed on the material by using 210 pairs of SSR markers evenly covering maize genome, and the population structure of these materials was revealed by Structure V2.3.4. Variance analysis of the average grain filling rate for different heterotic groups was also carried out to screen the lines with fast grain filling rate and know which group they belong to. 【Result】 Phenotypic analysis showed that the grain filling rate was very significantly different among different years, inbred lines, sampling times, lines×sampling time and lines×sampling time but repetition and year×lines at 0.01 levels. The correlation analysis showed that the grain filling rate on 10 and 20 days reached a extremely significantly positive correlation at 0.01 levels (0.515); and the 10 days grain filling rate with the 40 days grain filling rate and grain water content achieved significant negative correlation at 0.01 levels (-0.198, -0.228); at 0.05 levels, the 20 days grain filling with the 40 days grain moisture had notable negative correlation; the grain filling rate on 30 and 40 days with the 40 days cob moisture content, cob diameter, the 40 days grain water content, 30 and 40 days husk moisture content had extremely significant correlation at 0.01 levels; the correlation was found between 30 days and 10 days husk water content was significant; and the 40 days grain filling rate had very significant positive correlation with the 20 days husk water content. The population structure analysis showed that these maize inbred lines could be divided into 5 heterotic groups which are P, LRC, Reid, Lancaster and TangSPT groups. The variance analysis among the 5 heterotic groups showed that Lancaster and TangSPT had no significant difference at 0.05 level, the similar situation also emerged among the Reid, P and LRC groups, but Lancaster, TangSPT and Reid, P, LRC groups had significant difference. For multiple comparisons within each group between inbred lines, each group in inbred lines of grain filling rate at 0.05 levels had no significant difference. Thirty-three inbred lines with the filling rate higher than 0.8 g•100 grain-1•d-1 were screened in this study, and there are 13 lines in the Reid group, P, LRC, Lancaster, and TangSPT occupy 9, 6, 3, and 2 inbred lines, respectively. 【Conclusion】The grain filling rate showed significant differences among maize inbred lines, and it has different rhythm among the heterotic groups, P, LRC and Reid groups showed quick-quick-slow changing mode, while Lancaster and TangSPT groups showed quick-slow-slow changing rhythm.

Investigation of Sorghum Endosperm Cell Development and the Relationship with Its Maternal Tissue

LI Dong-liang, JING Yan-ping, LI Xiao-gang, GU Yun-jie, WANG Zhong

Scientia Agricultura Sinica. 2014, 47(17): 3336-3347. doi:10.3864/j.issn.0578-1752.2014.17.002

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【Objective】The objective of this research was to clarify the relationship between endosperm and its maternal tissue during development process of the sorghum caryopsis. 【Method】Sorghum KS-304 was used as the experimental material with its caryopsis development days precisely recorded and its caryopsis development closely observed. Structural changes of endosperm cells and relation with their maternal tissue were observed through semi-thin sections under light microscopy by applying Spurr resin tissue embedding. The ultrastructure of cells from different tissues and the starch granules wherein in developing and full ripe caryopsis were observed under SEM. The relationships of testa and aleurone layer of full ripe caryopsis were studied through fluorescent microscopy using cryosectioning.【Result】Four stages could be found in caryopsis development, they were formation stage, milky stage, dough stage, and full maturity stage. Accordingly, endosperm development was divided into five stages, i.e. coenocyte stage, cellurization stage, differentiation stage, developmental stage and the final maturation stage, and the first three of which are equal to that of the formation stage of caryopsis; while the last maturation stage corresponds to the last two stages of caryopsis development. It took as long as 15 days for the nucellar epidermis to be fully degraded. The outer peripheral endosperm cells started to accumulate lipid bodies as early as 7 DPA (days after pollination) and, turned into aleurone cells in 11 DAP. Only 1 layer of aleurone cells formed in full ripe caryopsis. Besides commonly seen aleurone granules and globoids, some single amyloplasts were also found on ripe aleurone cells, circa. 3 μm in diameter. Growth and development of endosperm cells also varied according to different locations, amyloplasts in cells surrounding embryo tended to have a slow growth rate and were loosely packed than other areas, making them floury endosperm compared to others’ corneous endosperms when finally matured. Amyloplasts constitution in subaleurone cells were quite composite, in a manner that mingles both aleurone and starchy endosperm cells. Amyloplast formation in starchy endosperm was unique, which has an “occurrence center” during initiation. Starch “grow” inside a tube-like plastid, and when mature, falls apart, leaving the mature amyloplast with an uneven oval shape. Starch/amyloplasts in mesocarp may not be exhausted approaching mature stage, and on the contrary, there seems to be a secondary growth in both quantity and diameter.【Conclusion】 Development of starchy endosperm in sorghum KS-304 showed a similar pattern to the corn. Starch formation of amyloplasts in starchy endosperm followed a unique way and is independent of all other crop spiecies. Cells of mesocarp layer may act as an extra “sink” during later stages in caryopsis development.

Cloning of Glutamine Synthetase BnGS2 Allele Genes from Ramie (Boehmeria nivea L.) and Study on Gene-Transforming Tobacco

ZHENG Jian-shu, YU Chun-ming, CHEN Ping, WANG Yan-zhou, TAN Long-tao, CHEN Ji-kang, ZHU Tao-tao, LU Ling-xiao, ZHU Juan-juan, DUAN Ye-hui, XIONG He-ping

Scientia Agricultura Sinica. 2014, 47(17): 3348-3358. doi:10.3864/j.issn.0578-1752.2014.17.003

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【Objective】 The objective of this paper is to clone and construct the over-expression vector of two glutamine synthetase (BnGS2) allele genes to study their effects on nitrogen metabolism of transgenic tobacco plants. 【Method】 The ramie transcriptome unigenes and RT-PCR were used to isolate BnGS2 allele genes which further identified by TaqⅠdigestion in self-bred F1 and parents. Sequence and structure were analyzed by bioinformatics. In addition, BnGS2 allele genes over-expression vector was constructed respectively according to homologous recombination technology and transformed through Agrobacterium tumefaciens LBA4404 using “leaf-disk” transformation method into Nicotiana tabacum. The transgenic T0 plants were verified by Kan screening and DNA PCR determination. The qRT-PCR was used for determining the relative expression levels of BnGS2 allele genes in T1 transgenic tobacco plants as well as the leaf GS activity, fresh weight, plant height, leaf soluble protein and total nitrogen content of transgenic plants were determined. 【Result】 Two BnGS2 allele genes with length of 1 340 bp containing 1 293 bp ORF region encoded polypeptide of 430 amino acids were isolated for the first time from ramie, named BnGS2-1 and BnGS2-2. The diversity of nucleotide in 11 sites among BnGS2 allele genes resulted in amino acid residues substitution at sites 195 and 382 (Pro-195 and Asp-382 in BnGS2-1, Thr-195 and Ser-382 in BnGS2-2). The NCBI Blastp analysis displayed that BnGS2 was close to Pisum sativum, Vigna radiate, Glycine max, Phaseolus vulgaris, and Medicago truncatula. In addition, the over-expression vectors of BnGS2-1 and BnGS2-2 were successfully constructed according to homologous recombination technology and the independent transgenic plants over-expressing BnGS2-1 and BnGS2-2 were obtained, respectively. Compared with wild type plants, the transgenic plants exhibited significant increase in leaf GS activity, fresh weight and leaf soluble protein content. The plant height and leaf total nitrogen content were slightly higher but not reached significant levels. However, these parameters between the transgenic plants with over-expression different BnGS2 allele gene (BnGS2-1 or BnGS2-2) did not exhibit significant difference. 【Conclusion】 Over-expression BnGS2 allele genes (BnGS2-1 and BnGS2-2) in the transgenic plants respectively resulted in a higher biomass and enhanced nitrogen use efficiency. In addition, there was no obvious difference in function between isolated BnGS2-1 and BnGS2-2.

Effects of Tillage and Straw Returning on Water Consumption Characteristics and Water Use Efficiency in the Winter Wheat and Summer Maize Rotation System

ZHAO Ya-li, XUE Zhi-wei, GUO Hai-bin, MU Xin-yuan, LI Chao-hai

Scientia Agricultura Sinica. 2014, 47(17): 3359-3371. doi:10.3864/j.issn.0578-1752.2014.17.004

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【Objective】Huang-Huai-Hai area is one of the most important areas that produce food crops. Frequent drought and flood are the main limiting factors for crop production, and the soil compaction, low topsoil and low water holding capacity are also the main factors causing the low and unstable yields of winter wheat and summer maize. Tillage and straw returning are two effective ways to reduce soil compaction, enhance water holding capacity and water use efficiency. The objective of the experiment was to study the effects of tillage, straw returning and their interaction on water consumption characteristics and water use efficiency in the winter wheat and summer maize rotation system.【Method】The experiment was conducted by a combination of different tillage ways and straw managements. Soil water consumption amount, soil water reduction amount, soil evaporation, grain yield and water use efficiency were analyzed by using six treatments which were conventional tillage with all straw returning, conventional tillage with no straw returning, deep tillage with all straw returning, deep tillage with no straw returning, subsoil tillage with all straw returning, subsoil tillage with no straw returning in the winter wheat and summer maize rotation system. The effects of tillage, straw returning and their interaction on water consumption characteristics and water use efficiency were analyzed.【Result】The results showed that, there were significant effects of tillage and straw returning on soil bulk density, soil water consumption amount, soil water reduction amount, soil evaporation, grain yield and water use efficiency. Compared with conventional tillage, deep tillage and subsoil tillage mainly decreased soil bulk density at 20-40 cm soil depth, increased the water consumption and soil water reduction amount at 0-100 cm soil depth of winter wheat and summer maize, while decreased the water consumption during fallow periods. Moreover, deep tillage and subsoil tillage also decreased soil evaporation during the growth period of summer maize. Deep tillage increased, but subsoil tillage decreased the soil evaporation during the growth period of winter wheat. Straw returning also decreased the soil bulk density, increased soil water reduction amount, increased soil water consumption amount during the growth period of winter wheat, but decreased the soil water consumption amount during summer maize growth period and fallow period. Moreover, straw returning increased soil water consumption, increased soil evaporation during the growth period of winter wheat, but decreased soil evaporation during the growth period of summer maize. Compared with the conventional tillage, the total grain yield of deep tillage and subsoil tillage increased by 10.7% and 9.8%, the water use efficiency increased by 8.8% and 6.3%. The total grain yield and water use efficiency of straw returning were 6.3% and 7.6% higher than the no straw returning treatment, respectively. A significant interaction between tillage system and straw returning was observed in soil water cosumption characteres, grain yield and water use efficiency of winter wheat and summer maize. Compared with conventional tillage with no straw returning, the total soil water consumption amounts of deep tillage with straw returning and subsoil tillage with straw returning increased by 3.3% and 2.4%, the soil water consumption amounts during the growth period of winter wheat and summer maize increased by 4.2% and 3.3%, while the soil water consumption amounts during the fallow period decreased by 7.0% and 9.9%. Moreover, the grain yields of deep tillage with straw returning and subsoil tillage with straw returning increased by 18.0% and 19.3%, the water use efficiency increased by 15.9% and 15.1%. 【Conclusion】In the six treatments, deep tillage with straw returning and subsoil tillage with straw returning showed the highest total grain yield and water use efficiency, and there was no significant difference in grain yield and water use efficiency between deep tillage with straw returning and subsoil tillage with straw returning. Therefore, it was concluded that deep tillage or subsoil tillage with straw returning is the most appropriate tillage practice in Huang-Huai-Hai area.

Adaptation Mechanism to Aquatic Environment of Cotton Seedlings Roots in Floating Nursing Seedlings in Nutrient Water-Bed (FNSNW)

ZHANG Hao, CHEN Jin-xiang, LIU Hai-he, ZHOU Zhong-hua, WANG Feng

Scientia Agricultura Sinica. 2014, 47(17): 3372-3381. doi:10.3864/j.issn.0578-1752.2014.17.005

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【Objective】Floating nursing seeding in nutrient water-bed (FNSNW) is a newly developed nursing seedling technique of cotton, but the adaptation mechanism to aquatic environment of roots is not clear. In this study, the possible mechanism of cotton roots adapting to FNSNW was analyzed using root structure, proteomics and the key gene expression.【Method】The matrix seedling method was used as control, ultrastructure and microstructure of main root tip of cotton seedling bred with FNSNW at different seedling stages were observed by transmission electron microscope and optical microscope. Protein difference between the control and aquatic root at the 3-leaf and 1-heart stage in FNSNW was analyzed using two-dimensional electrophoresis analysis. The expression of key genes including alcohol dehydrogenase gene and enolase gene for cotton seedling aquatic roots at different seedling stages and for different tissues of the same seeding at the 3-leaf and 1-heart stage in FNSNW was confirmed by real-time fluorescence quantitative PCR technology.【Result】In floating nursing seeding in nutrient water-bed (FNSNW), main roots of cotton seedling were found with appearance of aerenchyma. Compared with control, the cortical parenchyma cells of main root in FNSNW were smaller, and the growth of the xylem was weaker. Especially, the conduit diameter and the ratio of the conduit in stele were significantly smaller than the control. For tip cells of main root in FNSNW, there was phenol material at the 1-leaf and 1-heart stage, cortical cell gap at the 2-leaf and 1-heart stage, starch grain at the 3-leaf and 1-heart stage, and crystal cells with plasmolysis in the 4 leaf and 1-heart stage. As the protein two-dimensional electrophoresis results of aquatic roots and control roots at the 3-leaf and 1-heart stage, there were 28 differentially expressed proteins including 20 non-redundant proteins. Protein functional classification indicated that these proteins were related with metabolism, cell structure, cell defense, energy metabolism, protein synthesis and cell growth, and mainly involved in metabolic pathway including glycolysis, ethanol fermentation, and tricarboxylic acid cycle. Compared with control, the expression of alcohol dehydrogenase 2a gene of aquatic root in FNSNW was significantly different with first increase from the 2-leaf and 1-heart stage to the 4-leaf and 1-heart stage and then decrease from the 4-leaf and 1-heart stage to the 5-leaf and 1-heart stage, and the expression of enolase gene was significantly different with decrease from the 1-leaf and 1-heart stage to the 5-leaf and 1-heart stage. For the same cotton seedling in FNSNW at the 3-leaf and 1-heart stage, the relative expressions of these two genes of aquatic roots was significantly different from stem and leaf.【Conclusion】In FNSNW environment, seedlings adapt to the aquatic environment through changes in root structure and gene expression, which may be the adaption mechanism for cotton seedlings bred by FNSNW.

Regulation of Cultural Characteristics and Virulence in Verticillium dahliae Hyphal Type Strain V07DF2 by the Catalytic Subunit VdPKAC1 of cAMP-Dependent Protein Kinase A

DENG Sheng, ZHANG Xin, LIN Ling

Scientia Agricultura Sinica. 2014, 47(17): 3382-3391. doi:10.3864/j.issn.0578-1752.2014.17.006

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【Objective】The objective of this study is to investigate the roles of VdPKAC1, coding a catalytic subunit of cAMP-dependent protein kinase A, in the microsclerotial development, the conidia production and the virulence of Verticillium dahliae hyphal type strain. 【Method】Gateway® cloning technology from Invitrogen was used to construct the binary vector for the targeted deletion of VdPKAC1. In the T-DNA region of the vector, the 5′- and 3′-end homologous DNA sequences of VdPKAC1 were placed on the flank of the hygromycin resistance cassette. Mediated by the Agrobacterium tumefaciens AGL-1, this T-DNA was integrated into the genomic DNA of V07DF2, which is a highly aggressive defoliating V. dahliae strain and was isolated from the infected cotton in Jiangsu. Based on the PCR and the Southern blot analysis, five of six transformants with hygromycin resistance cassette were the targeted deletion mutants of VdPKAC1, and three of them were selected for further analysis. Seven days after cultured in PDB, the secretions of melanin was observed. Twenty-eight days after cultured on PDA plates, the growth of hypha and the formation of resting structure were investigated. Moreover, the ability of conidia production of mutants and wild type strain were assessed by the induction of cotton root extracts. And the virulence of the deletion mutants and wild type strain were also investigated. 【Result】Southern blot analysis indicated that the targeted deletion mutants 2B5 and C5 harbored a single insert of T-DNA, while in other mutants, ectopic integration were occurred. Compared with the wild type strain V07DF2, the mutants defective in VdPKAC1 produced more pigment in PDB, and formed chain-like resting structure which is different from normal microsclerotia. Moreover, the conidia production and pathogenicity of the mutants was reduced significantly against wild type strain V07DF2, but the surface of the mutant colonies was covered with more vigorous aerial hyphae. 【Conclusion】 The cAMP signaling pathway mediated by protein kinase A plays multiple roles in determining the traits of V. dahliae. It has been reported that VdPKAC1 (homolog VDAG_06474.1) is negatively regulated the formation of black microsclerotia in V. dahliae, its targeted deletion mutant produced more microsclerotia than wild type strain. To wild type strain V07DF2 which does not form any black microsclerotia or similar structure in the PDA plate, when the homologs gene of VdPKAC1 was knocked out, the targeted deletion mutant formed chain-like resting structure which is different from normal microsclerotia. The mutants with the novel phenotypes will be excellent materials for the identification of the functional genes involved in microsclerotia formation.

Molecular Basis of Resistance of Phytopathogenic Fungi to Several Site-Specific Fungicides

ZHAN Jia-sui;WU E-jiao; LIU Xi-li; CHEN Feng-ping;

Scientia Agricultura Sinica. 2014, 47(17): 3392-3404. doi:10.3864/j.issn.0578-1752.2014.17.007

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Site-specific fungicides play an important role in plant disease management. However, frequent applications of the fungicides over a large geographic scale can induce the emergence of resistant strains in the pathogen population. Resistance to fungicides with various modes of action has been documented in many plant fungal pathogens. This review summaries the current advances in understanding of the modes of action in five major classes of site-specific fungicides including methyl benzimidazole carbamate (MBCs), dicarboximide fungicides (DCFs), 14α-demethylase inhibitors (DMIs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) and the molecular mechanisms of resistance. Evolutionary process of fungicide resistance and management programme aiming to mitigate the emergence of resistance are also discussed in the review. The target protein of MBCs is β-tubulin, and the resistance in phytopathogenic fungi is linked to point mutation in the target protein. Amino acid substitutions in target protein occur mainly at the positions 50, 167, 198, 200, and 240, and the most frequent mutation is amino acid 198. In general, only one substitution occurs in each resistant isolate. Resistant level varies among isolates with different substitutions. The target protein of DCFs has been unknown, the resistance may be correlated with point mutation in histidine kindnase (OS-related) genes. DMIs inhibit sterol 14α-demethylation step in biosynthesis of ergosterol and resistant mechanisms usually include point mutation of Cyp51 or over-expressions of Cyp51 and transporter genes. But point mutation in Cyp51 is the major mechanism of DMI resistance. Different site mutations or even same site and same amino acid substitutions could lead to different resistance to triazoles. The number of point mutations in Cyp51 varies among fungi, ranging from one mutation to several mutations and different mutations have an additive effect on DMI-resistance. QoIs affect the electron transportation chain by binding to complex III and resistance in this type of fungicides is usually linked to point mutation in Cytb occurring usually at amino acids positions 120-155 and 255-280. The most frequent point mutations are G143A and F129L in Cytb. SDHIs inhibit complex II in electron transportation chain. Its resistance is generally related to point mutation either in SdhB, SdhC or SdhD, but in the majority of pathogens, resistance to SDHIs is due to point mutation in H272 of SdhB. In the contrast, the sites of point mutations in SdhC or SdhD vary among different pathogens.

Microfluidic Electrophoresis Detection of Tomato yellow leaf curl virus (TYLCV)

YU Ming-fen;ZENG Hong-mei; ZHONG Run-tao; ZHAO Xiao-ming; QIU De-wen;

Scientia Agricultura Sinica. 2014, 47(17): 3405-3413. doi:10.3864/j.issn.0578-1752.2014.17.008

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【Objective】The objectives of this study are to explore the effect of microfluidic electrophoresis on detection of PCR products, build a detection method for Tomato yellow leaf curl virus (TYLCV) by electrophoresis in microfluidic chip, and to remedy the defect in reagent consumption, long time, lack of safety of agarose gel electrophoresis.【Method】Primers in relatively stable positions of TYLCV genome were designed, some primers in references were taken into account, and these selected primers were verified. TYLCV primers were screened out based on the criterion of specificity, stability, and sensitivity. DNA standards φX174/BsuR I (Hae Ⅲ) marker was subjected to agarose gel electrophoresis and microfluidic electrophoresis, and the two methods were compared in supplies, time-consumption and sensitivity to confirm the value of microfluidic electrophoresis in nucleic acid detection. In order to evaluate the value of microfluidic electrophoresis in virus detection, the PCR amplification products of one pair of selected primers on the actual samples were processed by microfluidic electrophoresis.【Result】Fourteen pairs of TYLCV primers were screened out, 2 pairs came from the literatures and the other 12 pairs were designed in this study. Each pair of primers could meet the requirement for microfluidic detection. TYLCV-T was chosen from these primers for the subsequent study. By comparison of agarose gel electrophoresis and microfluidic electrophoresis, the time consumption and reagent consumption of microfluidic electrophoresis were 1/10 and 1/8 of those of agarose gel electrophoresis, respectively. The detection sensitivity of microfluidic electrophoresis was at least 103 times higher than that of agarose gel electrophoresis, which could detect accurately 5×10-6 μg?μL-1 of nucleic acid according to calculation of the DNA standards concentration. By comparing the microfluidic electrophoresis peak of the sample with that of DNA marker, the size of the nucleic acid could be determined.【Conclusion】Primers of TYLCV were screened, which can be used for further study of virus detection by microfluidic technology. Microfluidic electrophoresis was more dominant than agarose gel electrophoresis in sensitivity, effectiveness, reagent consumption and time saving. Microfluidic electrophoresis detection was initially established, which provides a new rapid detection approach for TYLCV.

Response of Rice Yield and Nitrogen Uptake to Enhanced Efficiency Nitrogen Fertilizer in China: A Meta-Analysis

YUAN Jun-li, LIANG Xin-qiang, LI Liang, YE Yu-shi, FU Chao-dong, SONG Qing-chuan

Scientia Agricultura Sinica. 2014, 47(17): 3414-3423. doi:10.3864/j.issn.0578-1752.2014.17.009

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【Objective】This study is the first to make a large-scale assessment of the effect of the application of enhanced efficiency nitrogen fertilizers (EENF) on rice yield and plant N uptake quantitatively to provide a scientific basis for evaluating the economic benefits of EENF and promoting the use of EENF in China.【Method】A total of 48 peer-reviewed studies were collected in Chinese and English books and journals to establish a field experimental database, and a meta-analysis was conducted to quantitatively evaluate the response of rice yield and N uptake to EENF in China and to determine under what conditions EENF are the most effective. 【Result】 The results suggested that, on average, the application of EENF made rice yield and N uptake increase by 7.5% (95% CI: 6.7%-8.4%) and 10.5%(95% CI: 9.5%-11.4%) , respectively. In terms of factors, it was found that soil pH had a significant effect on rice yield and N uptake, which was increased by 10.5% and 18.8% in alkaline soils(pH≥7.5) which are more than that in acidic (pH≤6.5) and neutral soils (pH: 6.5-7.5). Coated slow/controlled release nitrogen fertilizer (CRF) was better than nitrification inhibitors, especially for N uptake, nitrification inhibitor had no effect on it, but CRF made it increase by 17.9% than conventional fertilization; EENF applied as basal fertilization was better than split fertilization, which increased rice yield and N uptake by 4.2% and 7.5% and they were less than as basal fertilization. Besides, mixing EENF with control fertilizer had equal effect, and it was labor-saving; the best practice for N rate was 120-180 kg•hm-2, which increased rice production and N uptake by 6.5% and 12.1%, respectively. At last, the application of EENF in northern China was better than in southern China, because it increased rice yield and N uptake by 3.4% and 3.0% more than in southern China.【Conclusion】The best practice in this study to increase rice yield and plant N uptake is to use EENF, especially the coated slow/controlled release fertilizers (applied into alkaline soil as basal fertilizer) when the total N rate is 120-180 kg•hm-2.Application of nitrification inhibitors, especially 3,4-dimethylpyrazole phosphate (DMPP) is not suitable for paddy fields to increase N uptake in China. It seems that rice planting way (transplant or direct sowing) and fertilization methods of EENF (EENF only or mixed with conventional nitrogen fertilizers) have little difference in improving rice yield and N uptake. It may be better to apply EENF in northern China than southern China.

ffects of Long-Term Fertilization on Flue-Cured Tobacco Soil Nutrients and Microorganisms Community Structure

CHEN Dan-mei; DUAN Yu-qi; YANG Yu-hong; JIN Yan; HUANG Jian-guo; YUAN Ling

Scientia Agricultura Sinica. 2014, 47(17): 3424-3433. doi:10.3864/j.issn.0578-1752.2014.17.010

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【Objective】 Nutrients and microbes are the two most important indicators for soil fertility and productivity. The aim of the experiment is to study the relationship between fertilizer application for high soil fertility and fungal disease of crops so the lands can be used sustainably. A long-term fertilizer location experiment of tobacco was carried out in Yunnan Tobacco Agriculture Research Institute. Soil samples were analyzed for understanding the relationship between fertilization method, soil fertility and crop fungus diseases, scientific fertilization for flue-cured tobacco, maintenance of long-term sustainable development of agricultural production can be realized. 【Method】 The influences of long-term fertilization on organic matter (OM), nutrients and microbial community structure were studied by routine analysis of phosphorus lipid fatty acids (PLFAs), and 454 pyrosequencing with a 16-year tobacco grown field trial. The experimental treatments included blank control (without fertilizers, CK), chemical fertilizer (CF), and mixture of organic manure plus chemical fertilizer (MCF). 【Result】 After 16 years of fertilization, OM increased by 19.63% and available phosphorus increased in MCF treatment, while in CF OM decreased by 20.56% and alkali-hydrolyzable nitrogen and potassium decreased in soil. Fertilization, particularly MCF, also increased the type and total amount of PLFAs, indicating the increment of microbial groups and numbers. In CF treatment, however, the PLFAs proportion of fungi, the heterotrophic microbes, was 2.52 folds compared with MCF and fungal groups (optimizing taxon units, OTUs) were increased by 25.91% compared with MCF. The diversity, evenness and predominance indexes showed a similar trend of that of fungal proportion and groups. The results suggested the changes in soil ecosystem environment by CF that was beneficial to fungal reproduction and growth, resulted in more fungal groups, high intensity, and obvious predominance. In soil fungus community, all of the fungi belonged to Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota, and unidentified groups and most of them were Ascomycota. The abundance of the top 20 predominant fungi accounted for 33.01%-49.28%. Among them, 15 types existed simultaneously in soil in the treatments of MCF and CK, but only 6 in CF and CK. 【Conclusion】Therefore, a long-term chemical fertilization could be the reason for high fungal disease occurrence of crop but low organic matter in soil. Soil characteristics was one of the important factors for determining fungal communities, which could be changed slightly by MCF but greatly by CF for a long time.

The Toxicity Thresholds (ECx) of Cadmium (Cd) to Rice Cultivars as Determined by Root-Elongation Tests in Soils and Its Predicted Models

SONG Wen-en, CHEN Shi-bao

Scientia Agricultura Sinica. 2014, 47(17): 3434-3443. doi:10.3864/j.issn.0578-1752.2014.17.011

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【Objective】Although the attention has been paid for decades to the ecological risk of Cd to rice in soils, most studies focused on its healthy risk in terms of the food standard instead of its ecological risk and toxicity thresholds in soils. The toxicity thresholds (ECx, x=10, 50) and its predicted models of Cd to rice cultivars in various soils in China were determined using ISO 11269-1 root-elongation endpoints with the aim of providing fundamental data for the revision of soil environmental quality standards of Cd in soils of China.【Method】Eight different soils with various properties and three rice cultivars were selected in this study, the dose-response curves and the toxicity thresholds were investigated using Log-Logistic distribution models based on the ISO 11269-1 root-elongation test in soils, the predicted models for Cd toxicity was also developed in this study. 【Result】The results indicated that the relative root elongation (%) decreased with the Cd concentrations increment in soils. The 10% (EC10) and the half inhibiting concentration (EC50) of Cd to rice cultivars varied significantly among the tested soils and the rice species. In general, the toxicity thresholds (ECx, x=10, 50) of Cd to rice species decreased with it’s sensitiveness to Cd stress in soils, i.e. the ECx followed the order of T167＞L28＞X45. The significant differences of the toxicity thresholds (ECx, x=10, 50) of Cd to rice species were also observed in this study as determined with single rice species, e.g. in terms of the Cd-stress-sensitive cultivar of T167, the 10% inhibiting concentration (EC10) of Cd to root-elongation varied form 1.40-5.25 mg•kg-1 with the maximum variety of 275.0%, the half inhibiting concentration (EC50) varied form 17.83-46.93 mg•kg-1 with the maximum variety of 163.2%, respectively. However, for the Cd-stress-tolerance cultivar of X45, the 10% inhibiting concentration (EC10) of Cd to root-elongation varied form 1.72-8.22 mg•kg-1 with the maximum variety of 377.9%, the EC50 varied form 26.96-68.16 mg•kg-1 with the maximum variety of 152.8%, respectively. The multiple regression analysis showed that there was a significant positive correlation (P＜0.05) between soil pH, organic carbon (OC), cation exchange capacity (CEC) and the toxicity thresholds (ECx, x=10, 50) of Cd to rice cultivars in soils. A predicted model was developed and validated for the toxicity thresholds (ECx, x=10, 50) of Cd to rice with the measured ECx fell within the range of predicted values±2 standard deviations. 【Conclusion】 Significant difference of the toxicity thresholds (ECx, x=10, 50) of Cd to rice cultivars was observed in soils as determined by root-elongation test, and a significant positive correlation (P＜0.05) was also observed between soil pH, OC, CEC and the toxicity thresholds. The ECx of Cd toxicity were affected by the variance of the tested rice cultivars in soils, the ecological toxicity of Cd to different test endpoints should be considered for the future revision of the soil environmental quality standards of Cd in soils of China.

Isolation and Expression Analysis of PmKNAT2 Gene from Japanese Apricot

SUN Hai-long, SONG Juan, GAO Zhi-hong, NI Zhao-jun, ZHANG Zhen

Scientia Agricultura Sinica. 2014, 47(17): 3444-3452. doi:10.3864/j.issn.0578-1752.2014.17.012

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【Objective】 This paper aims to isolate the PmKNAT2 gene from Japanese apricot (Prunus mume Sieb. et Zucc.) cv ‘Daqiandi’, and analyze the structure and expression pattern of this gene, for further studying the molecular mechanism of Japanese apricot pistil abortion and molecular breeding. 【Method】 Specific primers were designed based on the peach sequence (EF093491) in NCBI, which is the highest homologue with peach gene KNOPE2. The improved CTAB method was used to isolate total RNA and the full length of PmKNAT2 cDNA was obtained by using RT-PCR and RACE. The sequencing data were assembled by DNAMAN software; BLASTn and BLASTp in NCBI were used to do the similarity analysis. PmKNAT2 gene structural characteristics were analyzed by the following software: DNAMAN was used to analyze the ORF and amino acid sequences and MEGA4.0 was used to create the phylogenetic tree; the protein molecular weight and isoelectric point were speculated by using Bioxm2.6; the conserved domain structure of protein was predicted by Conserved Domains program in NCBI; the protein secondary structure was predicted by using SOPMA program. The fusion expression vector PJIT166-PmKNAT2-GFP was constructed and then introduced into onion epidermal cells by the particle bombardment method; green fluorescence was monitored under a laser scanning confocal microscope. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of PmKNAT2 in different developmental stages of flower buds and different flower organs. 【Result】 The full length of PmKNAT2 cDNA was 1 402 bp and contained 47 bp 5′UTR, 293 bp 3′UTR and a 1 062 bp ORF which encoded a 353 amino acids polypeptide with a calculated molecular weight of 40.14 kD and an isoelectric point of 4.85. Protein structure analysis showed that PmKNAT2 contained two kinds of domain namely MEINOX area (KNOXⅠand KNOXⅡ) and HD area, which indicated that it belongs to the KNOX protein. Similarity analysis showed that the predictive amino acid sequence of PmKNAT2 compared with other sequences of KNOX in GenBank shared 50%-100% in homology. The phylogenetic tree analysis showed that PmKNAT2 was clustered together with peach KNOX protein, which was consistent with the morphological classification. In addition, the predictive secondary structure showed that PmKNAT2 protein was made up of 47.14% alpha-helix, 3.43% beta-turn, 3.14% extended strand and 46.29% random coil. Subcellular localization results showed that the PmKNAT2 protein localized in cell nucleus and cell membrane. Real-time PCR analysis showed that the expression level of PmKNAT2 was various in different stages of flower buds of ‘Daqiandi’ , the highest level in November. There were no significant difference in September, October and November. However, there was a significant difference between December and January. As for the determination of auxin content, the results showed that the highest level in January, and there was no significant difference in September, October and November; in contrast with the trend of gene expression. The expression analysis of flower buds in November showed that PmKNAT2 expressed in all the tissues, the expression level of imperfect flower (pistil brown, pistil deformity and no pistil) were higher than perfect flower. There was no tissue specific expression of PmKNAT2 gene between perfect flower and imperfect flower. The expression level in the sepal was higher than that in the stamen and the expression level in the stamen was higher than that in the petal. The lowest expression level of pistil was in perfect flower. 【Conclusion】 The abnormal expression of this gene might be related to pistil abortion in ‘Daqiandi’.

Difference in Starch Accumulation and Characterization of Sugar Metabolism During Fruit Development of Kiwi Fruit

ZHANG Hui-qin;XIE Ming; ZHANG Chen; YANG Lu-qiong; ZHANG Zhen;XIAO Jin-ping; ZHOU Li-qiu

Scientia Agricultura Sinica. 2014, 47(17): 3453-3464. doi:10.3864/j.issn.0578-1752.2014.17.013

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【Objective】The aim of this work is to study difference in starch accumulation between high- and low-starch- accumulating cultivars and characterization of sugar metabolism during fruit development, and to identify the related enzymes activities in order to provide a theoretical guidance for increasing fruit sugar content and improving flavour and quality of fruit.【Method】High-starch-accumulating variety ‘Hongyang’ and low-starch-accumulating variety ‘White’ were used to measure the changes in fruit relative growth rate (RGR), carbohydrate content (starch, glucose, fructose and sucrose), dry matter and enzyme activity in relation to carbohydrate metabolism throughout the growing season.【Result】The carbohydrate concentration was negatively correlated with fruit relative growth rate, and constantly increased with starch accumulation. The starch content was positively correlated with the soluble sugar contents (glucose, fructose and sucrose) and dry matter, and the ratios of maximum starch content to carbohydrates in ‘White’ and ‘Hongyang’ fruit were 87.1% and 84.1%, respectively. The higher rate of starch accumulation, which was also maintained for a longer period of time, was found in ‘Hongyang’, and the starch peak was much higher in ‘Hongyang’ than in ‘White’. The rate of starch accumulation and the maximum starch content in ‘Hongyang’ were 0.685 mg•g-1FW•d-1 and 70.78 mg•g-1FW, and they were 1.34 and 1.69 fold higher than those in ‘White’, respectively. The period of starch accumulation of ‘Hongyang’ was 21 days longer than that of ‘White’. When starch content in ‘White’ fruit rapidly dropped, starch accumulation of ‘Hongyang’ fruit linearly increased. As a consequence, the higher level of starch concentration in ‘Hongyang’ fruit maintained until harvest, while almost all starch in ‘White’ had been converted into soluble sugars prior to harvest. Neutral invertase (NI) and acid invertase (AI) activities were consistently much higher in ‘white’ than in ‘Hongyang’. In contrast, ADP-glucose pyrophosphorylase (AGPase) and sucrose phosphate synthase (SPS) activity was always stronger in ‘Hongyang’ than in ‘White’. Fruit starch content was positively and significantly correlated with AGPase and SPS activities, but was negatively and strongly correlated with NI and AI activities. NI activity was positively and significantly correlated with NI activity, while was negatively correlated with the activities of AGPase, SPS, sucrose synthase (SS), fructokinase (FK), glucokinase (GK) and UDP-glucose pyrophosphorylase (UGPase). AGPase activity in ‘Hongyang’ had positive and significant correlation with the activities of SPS, SS, FK, GK and UGPase.【Conclusion】Kiwi fruit accumulated carbohydrate predominantly as starch. AGPase was a determinant of high starch concentration in fruit, in addition to this, the variation of NI, AI and SPS activity may be the causes of high or low contents of starch, dry matter and soluble sugars in fruit.

The Regulation of pH Value of Liquid Feed on Blood Gas Parameters in Holstein Bull Calves

TU Yan;QIU Guo-liang; ZHOU Yi; YUN Qiang; QI Dong; WANG Jia-jie; DIAO Qi-yu

Scientia Agricultura Sinica. 2014, 47(17): 3465-3474. doi:10.3864/j.issn.0578-1752.2014.17.014

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【Objective】 Due to the immature gastrointestinal function and low ability digest dietary, diarrhea or other nutritional diseases were easy to arise in suckling calves that bring about low survival rate. Compared with adult cattle, liquid milk replacer is the main feed for the early weaned calves. Thus, acidity of milk replacer emulsions has more important effect on calf health. But the change regulation, the normal value scope of blood gas parameters in calves are not in any systematic manner, so that it is unable to determine whether the appropriate dietary acidity is. To study the change regulations of blood gas parameter in calves, an experiment was carried out to investigate the variations of blood gas parameters in calves which fed liquid feed with different pH. 【Method】The pH values of a milk replacer (6.2, 5.5, 5.0 or 4.5) and the ratio of vegetable protein to total protein in the milk replacer (50% or 80%) were used to form an 2 × 4 factorial design in this experiment. Forty-eight neonatal healthy Chinese Holstein male calves were allotted into eight groups and each group was fed with one of the 8 milk replacer emulsions. The experiment lasted 63 d with 21 d for adaptation and 42 d for test. Growth performance was determined fortnightly, the intake of milk replacers and pellet diet were recorded every day. Blood samples were collected on 21 d and 49 d. The pH value, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), oxygen saturation (SO2), the concentration of [HCO3-](HCO3-), actual base excess (ABE), total carbon dioxide volume (TCO2), standard bicarbonate concentration (SBC), standard base excess (SBE) of the blood samples were determined. Data of growth performance or blood gas parameters were analyzed using the GLM or MIXED procedure of SAS software, the regression equations between blood gas parameters and milk replacer emulsion pH value were established by REG procedure, and the changes of blood gas parameters were analyzed using normal distribution test. 【Result】The results showed that the average daily gain were improved while the pH value of milk replacer decreased (P＜0.05). The pH value of milk replacer siginificantly affected blood pO2, SO2, HCO3-, ABE, TCO2, SBC and SBE (P＜0.01). pH value, pCO2, pO2, SO2, HCO3- and TCO2 showed a quadratic curve (P＜0.05) as milk replacer emulsion pH value decreased with R2 as 0.9201, 0.8481, 0.8600, 0.9445, 0.8631, 0.8141, respectively, meanwhile ABE, SBC, SBE were showed a linear turn (P＜0.05) with R2 as 0.9060, 0.8126, 0.8298. Blood pH and pCO2 were varied remarkably with the age of day increasing (P＜0.05). Blood gas parameters were in line with the normal distribution (P＞0.05) except for HCO3-, ABE, SBE. While the ratio of vegetable protein to total protein of milk replacer increased from 50% to 80%, the average daily gain and dry matter intake of calves decreased, but blood gas parameters were unaffected (P＞0.05). 【Conclusion】The blood HCO3-, ABE, TCO2, SBC,and SBE of calves aged 21 to 63 days significantly decreased with the pH value of milk replacer dropping. Blood gas parameters can be used as sensitive indices to evaluate the effect of dietary acidity on calves.

Differences in Expression of HSPA2 in Different Tissues and Organs of Yak

WEN Ze-xing, YU Si-jiu, ZHAI Yu-jia, YANG Kun, LIU Peng-gang, HE Jun-feng, CUI Yan

Scientia Agricultura Sinica. 2014, 47(17): 3475-3482. doi:10.3864/j.issn.0578-1752.2014.17.015

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【Objective】 This experiment was conducted to study the differences in expression of heat shock protein 70-2 (HSPA2) in different tissues and organs of yak. 【Method】 Three 1-year-old Qinghai Plateau male yaks were used in the present study. Under normal physiological conditions, the tissues and organs were obtained from healthy yak in mid-september 2013 (heart, liver, spleen, lung, kidney, brain and testis). The RNA were extracted from different tissues and organs of yak, and the RNA were reverse-transcribed into first-strand cDNA. The primers were designed specifically according to the Bos grunniens HSPA2 and β-actin gene sequences (HSPA2:KC790105.1, β-actin: DQ838049.1). Firstly, RT-PCR were used to verify the real-time quantitative PCR (RT-qPCR) could be applied to the determination of the differences in expression of HSPA2 gene in different tissues and organs of yak. Then, real time quantitative PCR was applied to analyze the expression of HSPA2 gene. Paraformaldehyde was used to fix different tissues and organs of yak, and then made into paraffin sections. The protein localization of HSPA2 was measured by immunohistochemistry. The Image-Pro Plus 6.0 software was used to analyze immunohistochemistry, the integrated optical density of HSPA2 positive reaction was measured, and absorbance was analyzed. The SPSS19.0 software was used for significance of difference analysis. 【Result】 RT-PCR results showed that RT-qPCR can be applied to the determination of differences in expression of HSPA2 gene in different tissues and organs of yak. The results of RT-qPCR showed that comparative expression quantity of gene HSPA2 in testis was 83.33 fold than that in brain, 97.09 fold in kidney, 111.11 fold in heart,133.33 fold in spleen, 222.22 fold in lung and 285.71 fold in liver, respectively. Immunohistochemistry showed that HSPA2 expressed in the testis, kidney, brain, heart, lung, liver and spleen of yak. HSPA2 positive reaction was observed in yak renal cortical tubular, medullary tubules, cortical hippocampal CA1 region, cerebellar cortex, cardiac cells, alveolar epithelial cells, liver cells, spleen marginal zone and red pulp. The most positive reaction was found in the cytoplasm, and positive reaction was rarely found in the nucleus. In the testis seminiferous tubules, HSPA2 positive reaction was showed in both the cytoplasm and nucleus of spermatogenic cells. No positive reaction was observed in the negative control group. Analysis of integrated optical density values showed that the HSPA2 protein expression in testis was the strongest, and followed by the brain, kidney, heart, lung and liver, and its expression in spleen was the weakest. 【Conclusion】 By studying the gene and protein levels, the findings of the study showed the differences in expression of HSPA2 in different tissues and organs of yak. The expression of HSPA2 gene and protein in testis were higher than that in brain, kidney, heart, spleen, lung and liver, which suggests that HSPA2 may be associated with testicular reproductive function.

Transcription Profiles of Immune-Related Genes in Chickens Infected by Salmonella Enteritidis and Poly(I:C)

SUN Yan, LI Jian-chao, LI Peng, ZHENG Mai-qing, LIU Ran-ran, LI Qing-he, WEN Jie, ZHAO Gui-ping

Scientia Agricultura Sinica. 2014, 47(17): 3483-3491. doi:10.3864/j.issn.0578-1752.2014.17.016

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【Objective】 Genome-wide association studies were performed by using the chicken 60k high density SNP array for nine immune traits in Beijing-You chicken and identification of candidate genes and loci responsible for these traits were identified. Here, by treating Beijing-You chicken with polyinosinic acid-polycytidylic acid, Poly(I:C) and Salmonella enteritidis (SE), several candidate genes were further studied based on authors’ GWAS result, including CD1b, BMA1(B locus M alpha chain 1), TRIM27 (tripartite motif-containing 27) and ZNF692(zinc finger protein 692). 【Method】 Eighty 12-d-old Beijing-You Chickens were divided into three groups: the control group, Poly(I:C) treatment and SE treatment groups. All the experimental birds were reared in isolated facility. The treatment groups were, respectively, injected intramuscularly into the breast with 0.5 mL of Poly(I:C) and SE bacterial suspension containing 108 CFU and the control group was given 0.5 mL saline. Chickens were sacrificed at 12 h, 24 h, 3 and 6 days of post infection (DPI). Blood samples were collected and serum was stored. The bursa of Fabricius, thymus and spleen were rapidly removed to test inflammatory factors and gene expression. 【Result】 The weight was significantly lower than control group 24 h post infection (P＜0.01) and temperature of chickens was significantly changed in 24 h after treatments. In serum, concentrations of IFN-α, IL-4 and IL-6 were significantly higher than the control and reached a peak at 24 h or 3 d (P＜0.01). TNF-α increased in all periods and significantly higher than the control group after 3 days. The expression of candidate gene CD1b was not tissue-specific, but BMA1, TRIM27 and ZNF692 were highly expressed in thymus and bursa fabricius. In thymus, the expression of CD1b was significantly different between two treatments at 12 h and 24 h post infection. When treated with SE, the expression of CD1b was significantly increased and reached a peak at 24 h (P＜0.01). The mRNA expression of BMA1 was significantly different between two treatment groups in 12 h and 3 d post infection, when treated with Poly(I:C), the expression at 12 h post infection was lower than the SE group, but it was significantly higher than SE group (P＜0.01). The expression of TRIM27 on 6 DPI in Poly(I:C) treatment was significantly higher than control group, expression of ZNF692 was not different among three groups. In bursa of Fabricius, the expression of CD1b was the highest at 12 h after infection. There were no differences in the expression of genes BMA1 and TRIM27 between two treatments. The expression of ZNF692 was higher on 1 DPI in SE treatment than Poly(I:C), and also the highest on 3 DPI in both groups. 【Conclusion】 CD1b, BMA1,TRIM27 and ZNF692 might be involved in the immune response after treating with S. enteritidis and Poly(I:C). CD1b gene had an important role in the early stage of SE infections. BMA1 and ZNF692 were involved in immune response of thymus and bursa.

Construction of Genetic Map of Foxtail Millet (Setaria italica (L.) Beauv.) Using PCR-Based Molecular Markers

WANG Zhi-lan, WANG Jun, YUAN Feng, DU Xiao-fen, YANG Hui-qing, GUO Er-hu

Scientia Agricultura Sinica. 2014, 47(17): 3492-3500. doi:10.3864/j.issn.0578-1752.2014.17.017

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【Objective】 This study aims to construct a genetic map of foxtail millet (Setaria italica (L.) Beauv.) using PCR- based molecular markers. 【Method】 With 81 SSR markers distributed in nine chromosome of foxtail millet as reference markers, a total of 1 733 markers including SSR, STS, SNP, and SV from foxtail millet and ACGM and SSR from pearl millet, tall fescue and rice were used to screen polymorphic markers between Gao 146A and K103, and the polymorphic markers were tested in the F2 segregation populations derived from cross Gao 146A / K103. MAPMAKER VERSION 3.0 and MapDraw V2 were used for linkage analysis and integrated genetic map drawing.【Result】A genetic linkage map of foxtail millet containing 192 different kinds of markers was constructed, and 33 markers were newly mapped, of which 32 were from foxtail millet and 1 from pearl millet. The map consisted of nine linkage groups and covered the genome length 2 082.5 cM. Each linkage group was between 119.5-475.2 cM and the average distance of each linkage group spanned 231.39 cM. The number of markers on each linkage group ranged from 10 to 37 and the average distance between different markers was 10.85 cM. There were 36 markers accounted for 18.75% of 192 ones distributed in different linkage group and appeared segregation disorder. There appeared segregation disorder hotspots on LG2, LG6 and LG7, including 10, 15 and 7 segregation markers, respectively, and segregation disorder scattered on LG1, LG4, LG5 and LG8, however, no segregation disorder occurred on LG3 and LG9. According to transferability of molecular markers from different crops, there were 205 polymorphic markers of 1 235 ones from foxtail millet between parents and F2 population and the polymorphism rate was 16.60%, however there was only one polymorphic marker of 498 ones from pearl millet, tall fescue and rice.【Conclusion】A genetic linkage map of foxtail millet, covering the genome length 2 082.5 cM, was constructed using molecular markers from different species and most of the markers in this map were from foxtail millet.

Vacuolar Protein Sorting AtVPS25 Regulates Auxin Responses in Arabidopsis thaliana

GUO Meng-meng;CHEN Ming; LIU Rong-bang; MA You-zhi; LI Lian-cheng; XU Zhao-shi; ZHANG Xiao-hong;

Scientia Agricultura Sinica. 2014, 47(17): 3501-3512. doi:10.3864/j.issn.0578-1752.2014.17.018

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【Objective】The objective of this study is to identify the phenotype of homozygous mutant vps25 under auxin stress conditions, get the interacting protein of Arabidopsis vacuolar protein sorting AtVPS25, and analyze the function and molecular mechanisms of their interaction in the process of auxin response.【Method】The "three primer method" was adopted to identify the mutants. The function of the VPS like protein, AtVPS25, was analyzed by identification of the responses of AtVPS25 mutants to exogenous auxin. AtVPS25 protein was used as a bait to screen its interacting proteins by the split-ubiqutin system in Arabidopsis. The interaction between AtVPS25 and AtAIR12 (for Auxin - Induced Root in cultures) was confirmed by the yeast interaction experiment and the bimolecular fluorescence complementation test (BiFC). The subcellular locations of AtVPS25 and AtAIR12 protein was analyzed by the confocal scanning. The expression patterns of auxin transport-related genes in AtVPS25 mutants were identified by real-time PCR. The subcellular locations of AtVPS25 and AtAIR12 protein were analyzed in plants cells; The expression of part of auxin transport related genes in vps25 mutant under auxin treatment condition were identified by real-time PCR. 【Result】Real-time PCR results showed that under 10 μmol?L-1 IAA treatment conditions, in wild-type Arabidopsis thaliana (WT), the AtVPS25 expression level was increased with the stress time, and reached the highest at 12 h , about 40 times the size of 0 h, which proved that AtVPS25 was induced by auxin treatment. The AtVPS25 protein was used as a bait to screen the interacting protein from Arabidopsis by using the split-ubiqutin system and get the interacting protein AtAIR12. Two-hybrid interaction tests of AtVPS25 and AtAIR12 protein full-length sequence proved that AtVPS25 interacted with AtAIR12. Subcellular localization test proved that AtVPS25 is located on the cell membrane and cytoplasm, AtAIR12 is located on the cell membrane and chloroplast membrane. BiFC (bimolecular fluorescence complementation) test results showed that AtVPS25 is interacted with AtAIR12, and the interaction sites were on the cell membrane and cytoplasm. Molecular identification showed that homozygous mutant vps25 was obtained. When vps25 growth in conditions of 0.1 mg?L-1 IAA, the elongation of primary root was inhibited, and the difference in primary root was relatively significant (P＜0.01) compared with WT in the same condition, while no significant difference in the number of lateral roots, which have been reported the similar phenotype of the mutant air12-1 under the stress conditions of auxin. When treated with 10 μmol•L-1 IAA, under WT background conditions, the expression level of AtAIR12 responsed obviously for IAA, and increased with the stress time, reached a maximum at 12 h, about 80 times as 0 h, which proved that AtAIR12 and AtVPS25 have the exactly same change trend in WT under conditions of 10 μmol•L-1 IAA treatment. Under the background of vps25 mutant, the expression of AtAIR12 was restrained relative to the WT, the expression level had no obvious change from 0 to 24 h. Additionally, the expression level of auxin efflux carrier gene (AtPIN2) was reduced, while the auxin input vector gene was increased in mutant vps25.【Conclusion】 IAA had the effects of inducing the expression of vacuolar sorting protein gene AtVPS25 in Arabidopsis and AtVPS25 regulated the development of plant primary root. AtVPS25, which interacted with AtAIR12 at cell membrane and cytoplasm, regulated the expression of some IAA associated genes. Results indicated that AtVPS25 had influence on the response of IAA in plant root through regulating expression level of downstream genes above-mentioned. Further research is required to clarify the regulatory mechanism between AtVPS25 and AtAIR12 in plants.

Creation and Chromosome FISH Identification of Cucumber Materials with Different Ploidies

GUAN Wei, ZHANG Yun-xia, YANG Shu-qiong, CHEN Jin-feng, LOU Qun-feng

Scientia Agricultura Sinica. 2014, 47(17): 3513-3522. doi:10.3864/j.issn.0578-1752.2014.17.019

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【Objective】 The narrow genetic basis and limited genetic diversity of germplasm resources were the main bottlenecks of genetic and breeding researches of cucumber (Cucumis sativus L.). This research aimed to create different euploidy and aneuploidy cucumber germplasm materials, and to establish a reliable method for identifying chromosomal constitution of the new materials, which would lay a foundation for the researches of screening chromosome lines, chromosome localization and genetic breeding. 【Method】 The germinating seeds of the North China ecotype inbred line ‘Changchunmici’ of C. sativus were treated with 0.4% colchicine solution to induce the chromosome doubling. Autotriploid was obtained through culturing the 35-45 d zygotic embryo from the cross of the induced autotetraploid and diploid. The chromosomal ploidy or number of the induced plants and hybrids were investigated using chromosome counting, combined with the morphology, leaf stomata under electron microscope scanning. Fluorescence in situ hybridization (FISH) was carried out to ascertain the chromosomal constitution of the induced plants based on the number, intensity and location of specific probe signals. 【Result】 According to the ploidy identification based on the mitotic metaphase chromosome number, eight autotetraploidy plants (2n=28) and three aneuploidy plants (2n=16, 19, or 27) were obtained in this study. Autotriploid plants (2n=21) were produced from the cross of autotetraploid and diploid. FISH signals of Type III (cucumber centromere probe) and 45S rDNA were multiple changes among diploid, triploid and tetraploid. The result further indicated the ploidy level. There were differences in morphologic characteristics among different ploidy plants of ‘Changchunmici’. Compared with diploid, tetraploid had significant differences in morphologic characteristics. The difference among triploid, aneuploidy and diploid were not significant, but the aneuploid grew weaker and with late flowering period and low fruit setting rate. Results from leaf stomata under electron microscope scanning showed that there were differences among different ploidy plants. With the increase of ploidy, the length and width of leaf stomata rose notably, but the stomatal density declined obviously, which could be used as an aid for identifying the ploidy of cucumber. The tandem repetitive sequence (45S rDNA and Type III) and chromosome-specific single copy gene Csa006700 were used as FISH probes to identify chromosomal constitution of the aneuploidy plant with the chromosome number of 16. FISH results from repetitive probes on the mitotic metaphase chromosomes showed that the extra two chromosomes were chromosomes 1 or 2. Further, the chromosome 2 specific gene probe-Csa006700 showed the signals on one pair of chromosomes. These results confirmed that this aneuploidy plant was assigned as tetrasome polyploidy with two extra chromosome 1 of cucumber (2n=14+2). These results confirmed that colchicine could directly not only induce autopolyploid, but also induce a variety of aneuploid. 【Conclusion】 It is a rapid method to create cucumber different euploidy and aneuploidy germplasm materials using colchicine, and the chromosomal constitution could be identified by FISH analysis based on the chromosome specific probes.

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