Scientia Agricultura Sinica

Heterosis loci and QTL of Super Hybrid Rice Liangyoupeijiu Yield by Using Molecular Marker

XIN Ye-Yun-1, 2 , YUAN Long-Ping-1, 2

Scientia Agricultura Sinica. 2014, 47(14): 2699-2714. doi:10.3864/j.issn.0578-1752.2014.14.001

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【Objective】 The heterosis loci and QTLs of yield and yield components were detected by using a RILsBCF1 population derived from a cross between Pei’ai 64S and 9311. The relationship was explored between the genetic variance of these two parental lines and yield heterosis in the resulted hybrid for predicting hybrid heterosis.【Method】Based on a population of 219 recombinant inbred lines (RILs) of F8 generation produced by single seed descendant method from the Pei’ai 64S×9311 cross, a RILsBCF1 population was generated by backcrossing of RILs to Pei’ai 64S. With a total of 151 polymorphic SSR markers, a linkage map was constructed spanning 1 617.7 cM across the whole genome with an average marker interval of 10.93 cM. The correlation between genetic distances and F1 trait performance of RILsBCF1 and their prediction in yield and yield component traits were conducted respectively by using molecular marker analysis, one-way ANOVA with different freedoms, and composite interval mapping using mix linear model in SAS, together with heterosis prediction and QTL mapping. 【Result】The RILsBCF1 used in the study showed significant diversity with high segregation in multiple traits, and their average performance was significantly higher than that of RILs F8. In this RILsBCF1 population, 74 heterosis positive loci and effect-increasing loci were identified by two-group method and three-group method in yield and yield component traits, respectively. Compared with two-group method, three-group method could get more positive loci or effect-increasing loci to a certain degree and raise efficiency of predicting correlationship between genetic distances of both positive loci and effect-increasing loci and F1 traits’ performances. The result of the effect-increasing loci detecting was the same in both two-group and three-group methods. Six heterosis loci were detected at the same regions for three traits (Sterile lemma per panicle, Grains per panicle and Pencentage seed setting), overlapped with three yield effect-increasing loci clustered on chromosome 7. Based on the relationship between marker-effect values of yield effect-increasing loci using the three-group method and F1 trait performance, four multiple regression prediction models were constructed using a stepwise procedure. A total of 28 markers with heterozygous genotypes were identified to significantly increase the correlation coefficient between the genetic distances and the F1 trait performance from 0.335 to 0.617. Three QTLs for yield heterosis and three QTLs for grain per panicle heterosis were mapped using this RILsBCF1 population. The mapped loci of QTL QGpp7 for grain per panicle heterosis and QHy7 for yield heterosis matched the effect-increasing loci identified by the methods of two-group and three group analyses. 【Conclusion】The approaches of screening more positive loci or effect-increasing loci and specific markers which influent heterosis can increase the correlation coefficient between the genetic distances and the F1 traits performances, and thus can be applied more efficiently in predicting the yield heterosis of rice hybrids with genetic distance of molecular markers. The yield QTL QHy7 located on chromosome 7 with a yield increase contribution of 7.48% can be used for yield heterosis prediction and in hybrid rice breeding. A heading stage QTL located between RM293-RM468 on chromosome 3 with the contribution of 14.9% can be used for rareripe high yield rice breeding.

Detection of GUS Protein and Its Expression Pattern in Transgenic Rice Plants

NIU Dong-Dong-1, HAO Yu-Jie-2, RONG Rui-Juan-1, WEI Han-Fu-2, LAN Jin-苹1、Shi-Jia-Nan-1, WEI Jian-1, LI Xue-Jiao-1, YANG Shuo-1, XI Wen-Hui-2, WU Peng-Cheng-2, LIU Li-Juan-1, WU Lin-3, LIU Si-Qi-3, YIN Chang-Cheng-2, LIU Guo-Zhen-1

Scientia Agricultura Sinica. 2014, 47(14): 2715-2722. doi:10.3864/j.issn.0578-1752.2014.14.002

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【Objective】 The objective of the current research is to establish a method for detecting GUS protein in transgenic rice via immunoassay and reveal the expression pattern of GUS protein driven by cauliflower mosaic virus (CaMV) 35S promoter.【Method】Genomic DNA isolated from E. coli was used as template to amplify Gus gene, the PCR product was cloned into pET30a vector, sequencing verified recombinant was transferred into E. coli expression strain BL21, recombinant protein was induced by adding IPTG and His-tag beads purified recombinant protein was used as immunogen to generate monoclonal antibody. The GUS protein-specific antibody was selected using western blot analysis. Broadford assay was used to quantify recombinant GUS protein, a standard curve of GUS protein concentration versus the signal intensity of western blot analysis was generated and the quantification of GUS protein in rice leaves was carried out by comparing its intensity with the standard curve. Total proteins were isolated from different tissues at different developmental stages, including shoot and root at seedling stage, stem, node sheath, cushion, upper, middle and lower part of leaves at tillering stage, stem, stalk, sheath, cushion, leaves, spike (1 cm, 2 cm, 10 cm and 20 cm in length, respectively) at booting state, stem, stalk, sheath, leaves and spike at flowering stage, stem, leaves, seed at different days after pollination (10 d, 20 d, 30 d and 40 d, respectively) at mature stage, embryo, endosperm and lemma at milky mature stage, whole seed, embryo, endosperm and lemma of mature seed, leaves and roots at different developmental stages. SDS-PAGE separated total proteins were detected by GUS-specific antibody to detect the abundance of GUS protein.【Result】An anti-GUS monoclonal antibody with high specificity was obtained (clone number #27), specific band was detected by western blot analysis using the antibody for GUS protein in transgenic rice or recombinant GUS protein, while no visible background signal could be detected. The lower limit for recombinant GUS protein detection was about 4 ng, and GUS protein could be detected in about 2.5% single grain sample of transgenic rice. The abundance of GUS protein in transgenic rice leaves at different stages was pretty stable, while its abundance in roots was decreased dramatically, specifically, the abundance of GUS protein in 5-leaf stage was one third less than in that at 3-leaf stage, and it was undetectable at 6-leaf stage. GUS protein accounted for about 0.02 ‰ of the fresh weight in rice leave at seedling stage. Further more, GUS protein was constitutively expressed in almost all tested samples in transgenic rice except the roots after tillering stage. However, slight different in abundance was detected among tissues, e. g. the abundance in stems and lemmas at booting and flowering stages was lower than that in leaves.【Conclusion】A applicable immunoassay method was established for GUS protein detection in transgenic rice, which is specific to the abundance of GUS protein while independent of enzymatic activity, about 0.6 mg sample is needed for the detection and results obtained from different laboratories using same reference protein can be compared. The constitutive expression of GUS protein driven by CaMV 35S promoter in transgenic rice was demonstrated.

The Trend of Quality Traits of Maize Varieties Released Extensively in Different Eras in China

SUN Qi-1, 2 , ZHANG Shi-Huang-1, LI Xin-Hai-1, MENG Zhao-Dong-2, CI Xiao-Ke-1, ZHANG De-Gui-1, HAO Zhuan-Fang-1, WENG Jian-Feng-1, BAI Li-1, LI Ming-Shun-1

Scientia Agricultura Sinica. 2014, 47(14): 2723-2730. doi:10.3864/j.issn.0578-1752.2014.14.003

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【Objective】 The experiment materials were the varieties released from 1950 with large area application in the production. The trend of quality traits of maize varieties released extensively in different eras in China was studied. The result will be useful for guiding the genetic improvement of quality traits of maize.【Method】Thirty-five cultivars extended in 1950-2000 at large scale were selected. All the varieties were planted in the Shunyi experimental field of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Each cultivar was sowed for 5 rows, 3 replications, randomized block design. Two rows in the middle of each plot were reaped. The grain yield and the volume-weight were observed after harvest. The contents of starch, protein and fat of seeds were measured, respectively, with near infrared reflectance spectroscopy (NIRS). ANOVA analysis was made for the quality traits and grain yield. The correlation between the quality traits and grain yield was analyzed. Base on the regression analysis on the mean value of quality traits and yield, the trend of them was studied. 【Result】 Within each decade, the coefficient of variance (CV) was below 10% for the contents of starch and protein and the volume-weight of seed. But the coefficient of variance (CV) was 9.54%-18.74% for the content of fat of seed and 4.53%-33.33% for the grain yield. It meant that the variance range within each decade was small for the content of starch, protein and the volume-weight of seed, but large for the content of fat of seed and the grain yield. The difference of the grain yield between decades was significant, the same as the contents of starch, protein and the volume-weight of seed. The difference in content of fat of seed was not significant. The grain yield of the maize cultivars released extensively increased gradually from 1950 to 2000, average 871 kg•hm-2 per ten years. The content of starch of seed increased generally with the decades, 0.25% per ten years in average. But the content of protein of seed decreased gradually with the decades, 0.31% per ten years in average. The volume-weight also increased generally with the decades, 5.97g•L-1 per ten years in average. The relationship between the content of starch of seed and the grain yield was significantly positive correlation (r=0.493). But the relationship between the content of protein of seed and the grain yield was significantly negative correlation (r=-0.678). It showed that the increase of the starch content and the decrease of the protein content of seeds from 1950 to 2000 was related to the improvement of yield.【conclusion】The grain yield of the maize varieties released extensively was improved greatly from 1950 to 2000. The content of starch and the volume-weight of seed were improved, but the content of protein of seed decreased. The fat content of seed had no obvious changes.

Advances in Research on Crop Yield Gaps

YANG Xiao-Guang, LIU Zhi-Juan-

Scientia Agricultura Sinica. 2014, 47(14): 2731-2741. doi:10.3864/j.issn.0578-1752.2014.14.004

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Demand for food is quickly rising with increases in population and living standards. In the past few decades, crop yields increased rapidly due to the utilization of rice seedlings and mulching technologies, improvement of managements such as irrigation and fertilizer, new varieties selections and technology improvements. However, yields in farmer fields are much lower than potential yields, which have been widespread in the world's agricultural production. Therefore, closing the gap between current and potential yields is important to increase the crop yield and make sure the food security. In this study, the definition, research method, and the main results of yield gaps were reviewed. Furthermore, some prospects of yield gaps in the future were made, which will provide reference for further research on yield gaps. Until now there are many different definitions to yield gap, however, in general the maximum yield is the potential yield, and total yield gap is the difference between actual and potential yield. The yield gap caused by a variety of factors, including no-transferable technology and environment constraints, biological constraints (variety, diseases and insects, etc.), and socio-economic constraints (cost and returns, policy, knowledge, and tradition, etc.). In order to analyze the yield gap in detail, scholars divided the yield gap into different levels according to their objectives. There are two kinds of research methods for yield gap, the survey and statistical analysis methods, and crop simulation models method. The survey and statistical analysis methods have a simple concept and easy to be operated, but requires sufficient experiment data, which the cost is higher and the duration is longer; the crop simulation models method can design more scenarios using the computer, but can not quantify all of the management accurately. Therefore, in the yield gap researches, we should combine the statistical methods, crop simulation models and remote sensing technology should be combined for taking the advantages of each method. A comparison of the crop yield gap around the world indicates that for the developed countries, potential ascension of crop production is smaller due to the relatively higher levels of cultivation management. There are many studies on the yield gaps for crops around the world, which provide a scientific basis for enhancing the crop yield and closing the yield gap. However, there are large differences between their results because of different methods used. Due to the limitations of data and methods, most researches have been focused on the constraints of climate, soil, variety, and cultivation management factors on the yield in agricultural production, but ignored the wishes of farmers, policy and economic factors. Therefore, a subsequent study of crop yield gap should quantify the potential yield of the main crops in each region, and identify the constraints of climate, soil, variety, cultivation management, and socio-economic factors on the yield in agricultural production.

Effects of Interaction of N and P on Rice Canopy Spectral Reflectance and Its PNN Identification

LI Ying-1, XUE Li-Hong-2, PAN Fu-Yan-1, YANG Lin-Zhang-2

Scientia Agricultura Sinica. 2014, 47(14): 2742-2750. doi:10.3864/j.issn.0578-1752.2014.14.005

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【Objective】Nitrogen (N) and phosphorus (P) are macronutrients for crops and the diagnosis of N and P in crops is the premise of scientific fertilization. Accurate, fast, and nondestructive detection of the deficiency of N and P in rice has a great meaning on precision fertilization, cost-saving and agricultural non-point source pollution control.【Method】A two-factor pot experiment of N (6 levels) and P (2 levels) was carried out, canopy spectral reflectance and plant TN and TP content were measured simultaneously at tillering, jointing and heading stages. The interactive effects of N and P on rice growth (N and P content) and canopy reflectance at 350-1 330 nm was investigated and PNN model was used to classify the N and P levels based on the canopy reflectance. In order to avoid the error of different batches caused by instrument, light, wind, temperature, water and other environmental conditions, the reflectance spectra data were standardized. A total of 2/3 of the data were used to train the PNN model and the other 1/3 data were used to test the PNN model. 【Result】 Rice N content was significantly influenced by the N rate, P rate and the interaction of N and P. But rice P content was only affected by P rate and N rate, the interaction of N and P did not exist. The response of canopy reflectance spectra to N rate was not influenced by P rates, and N deficiency increased the reflectance at visible band and decreased those in the near infrared region. Under P-deficiency, the reflectance at near-infrared bands decreased at all N levels, but the reflectance at visible bands increased in N application treatments while declined at tillering stage, increased at jointing stage, then decreased at heading stage when the N was seriously deficient. The identification accuracy of PNN model was highest at jointing stage and lowest at heading stage. The identification accuracy at tillering and jointing stages was 83% and 94% for P levels, and 78% and 88% for N and P deficiency levels, respectively. However, the identification accuracy for N levels and interaction of N-P levels was only 61%-75%. It was noted that all the N-deficient treatments were not identified as P-deficiency and vice versa at tillering and jointing stages, which showed that PNN model could distinguish N-deficiency from P-deficiency.【Conclusion】Rice canopy reflectance was influenced by N and P fertilizer levels. The PNN model of the rice canopy reflectance can not only identify nitrogen and phosphorus fertilizer levels based on single factor, but also can distinguish N-deficiency from P-deficiency, and has important meaning and value in rice fertilization decision and may avoid yield and economic loss and environmental pollution caused by improper fertilization strategy.

Effects of Supplemental Irrigation Based on the Measurement of Moisture Content in Soil Layers at Jointing and Anthesis Stages on the Chloroplast Ultramicrostructure and Chlorophyll Fluorescence Parameters of Flag Leaves of Winter Wheat

LIU Yue-Lan, YU Zhen-Wen, ZHANG Yong-Li, SHI Yu, WANG Dong-

Scientia Agricultura Sinica. 2014, 47(14): 2751-2761. doi:10.3864/j.issn.0578-1752.2014.14.006

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【Objective】 The field experiment was carried out in 2011-2012 and 2012-2013 wheat growth seasons. Four soil layers (0-20, 0-40, 0-60 and 0-140 cm) were designed to make the supplemental irrigation at jointing stage and anthesis stage (target soil relative moisture content=75%), no irrigation during the whole growth season was used as the control. The objective of this experiment was to study the effects of supplemental irrigation based on the measurement of moisture content in different soil layers at jointing stage and anthesis stage on the chloroplast ultramicrostructure and chlorophyll fluorescence parameters of flag leaves of winter wheat, so as to provide a theoretical basis and technical reference for water-saving and high-yield cultivation of wheat. 【Method】 The research was carried out with wheat cultivar Jimai 22, transmission electron microscopy (TEM) was used to analyze the chloroplas ultramicrostructure of flag leaves, the alcohol extraction method was used to analyze the chlorophyll content of flag leaves and chlorophyll fluorometer was used to analyze the chlorophyll fluorescence parameters of flag leaves. This experiment studied the effects of different treatments on the chlorophyll content, the chloroplas ultramicrostructure and the chlorophyll fluorescence parameters of flag leaves, grain yield, water use efficiency and economic benefit of wheat.【Result】When making supplemental irrigation in 0-40 cm soil layer, the oval chloroplasts of flag leaves with intact membrane closely arranged along the cell membrane on day 22 after anthesis. Clear grana lamellae arranged along the long axis of chloroplasts. And clear stroma lamellaes connected grana lamellas. The obvious damages of chloroplas ultramicrostructure were observed when making supplemental irrigation in 0-20 cm soil layer, and the chloroplas ultramicrostructure was caused more serious damage when no irrigation during the whole growth period. The round chloroplasts of flag leaves with damaged membrane arranged disordered and cell wall had a breakdown. The chloroplast ultramicrostructure had no significant difference between supplemental irrigation in 0-60 cm soil layer and 0-40cm soil layer. When making supplemental irrigation in 0-140 cm soil layer, damages of chloroplast ultramicrostructure were observed. Chloroplasts membranes were intact, membranes of cells was damaged and there were gaps among grana lamellas. There were significantly positive correlation among the numbers of chloroplast, chloroplast grana, grana lamellae of mesophyll cell of flag leaves and chlorophyll content (r＝0.99**, 0.99**, 0.96**). The numbers of chloroplast, chloroplast grana and grana lamellae of mesophyll cell under supplemental irrigation in 0-40 cm soil layer significantly increased than those under supplemental irrigation in 0-20 cm soil layer and no irrigation during the whole growth period, and which was why there was high chlorophyll content under supplemental irrigation in 0-40 cm soil layer. When making supplemental irrigation in 0-60 cm and 0-140 cm soil layers, the numbers of chloroplast, chloroplast grana and grana lamellae of mesophyll cell had no significant increase and so as to chlorophyll content. The maximal quantum yield of PSII (Fv/Fm), actual efficiency of PSII (ΦPSII) and photosynthetic electron transport rate (ETR) of flag leaves, 1000-grain weight, grain yield, water use efficiency and economic benefit of wheat under supplemental irrigation in 0-40 cm soil layer significantly increased than those under supplemental irrigation in 0-20 cm soil layer. When making supplemental irrigation in 0-60 cm and 0-140 cm soil layers, Fv/Fm, ΦPSII and ETR had no significant increase, so as the 1000-grain weight, grain yield, water use efficiency and economic benefit of wheat. 【Conclusion】The most important factor for higher 1 000-grain weight and grain yield was supplemental irrigation based on soil moisture measurement in 0-40 cm soil layer which could maintain better chloroplast ultramicrostructure, higher numbers of chloroplast, chloroplast grana, grana lamellae of mesophyll cell of flag leaves, higher chlorophyll content and higher chlorophyll fluorescence parameters during the middle late grain filling stage. Supplemental irrigation based on soil moisture measurement in 0-40 cm soil layer was the most appropriate irrigation treatment for recommendation by considering grain yield, water use efficiency and economic benefit of wheat.

Biological Characteristics and Genetic Diversity of Ustilaginoidea virens from Rice Regions in China

WANG Wen-Bin-1, 2 , ZHANG Rong-Sheng-2, LUO Chu-Ping-2, YIN Xiao-Le-2, LIU Yong-Feng-2, LU Fan-1, 2 , CHEN Zhi-Yi-1, 2

Scientia Agricultura Sinica. 2014, 47(14): 2762-2773. doi:10.3864/j.issn.0578-1752.2014.14.007

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【Objective】The objective of this study is to reveal the relationship among geographical regions, biological characteristics and genetic diversity of 111 strains of Ustilaginoidea virens from 10 provinces in China.【Method】Sporulation, growth rate and pathogenicity of these strains were evaluated by biological methods, and the relativity was analyzed by using the software SPSS 20.0. DNA was extracted from the strains, the genomic fingerprint profiles of 111 strains were generated by BOX-PCR, REP-PCR and ERIC-PCR, respectively. Their genetic diversities were further investigated by using clustering analysis.【Result】The correlation coefficient of sporulation and pathogenicity was 0.765 and the significance probability was 0.001, the correlation coefficient of growth rate and pathogenicity was 0.035 and the significance probability was 0.685. The genomic fingerprint profiles of 111 strains were generated by BOX-PCR, REP-PCR and ERIC-PCR, respectively. The genetic diversities of the U. virens population were 0.73 (in BOX), 0.62 (in ERIC) and 0.60 (in REP), respectively. The number of polymorphic bands was between 7 and 13 of each examined isolate detected through BOX-PCR analysis. According to cluster analysis of UPGMA, 6 clusters were grouped based on the boundary level of 0.70 average distances. The predominant group was group one which included 57 strains (51.4%). The strains of U. virens from Anhui, Sichuan, Guangxi, Hubei and Yunnan were grouped into group 1 and group 4. The strains of group 2 were mainly from Jiangsu, including 18 strains (16.2%), the strains of group 3 were from Zhejiang, including 8 strains (7.2%), the strains of group 5 were from Heilongjiang, including 9 strains (8.1%), and the strains of group 6 were from Liaoning, including 12 strains (10.8%). There was some correlation between BOX groups and geographic regions, no relationships were observed between BOX groups and their biological characteristics.【Conclusion】The relativity between the sporulation and pathogenicity was assumed to be medium. There was no relativity between the growth rate and pathogenicity. There was a significant relationship between BOX groups and the geographical regions, these results suggested that, naturally, U. virens in China might not be spread over long distances. No significant relationships were observed between BOX groups and their biological characteristics.

Expression and Analysis of β-1, 3-Glucanase Gene in Wheat TcLr35 Induced by Wheat Leaf Rust Pathogen and Signal Molecule

LI Xiao-Ying, GAO Lin, ZHANG Yan-Jun, WANG Hai-Yan, LIU Da-Qun

Scientia Agricultura Sinica. 2014, 47(14): 2774-2783. doi:10.3864/j.issn.0578-1752.2014.14.008

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【Objective】β-1, 3-glucanase, a kind of pathogenesis-related protein, plays an important role in signal transduction in plants. The objective of this study is to understand the expression patterns of β-1, 3-glucanase gene in the interaction between Puccinia triticina and wheat TcLr35, identify the relativity between the aim gene and resistance to leaf rust pathogen attack, and explore the molecular mechanism of PRs mediated resistance in adult wheat to P. triticina.【Method】Based on the previous studies, the structure characteristics of β-1, 3-glucanase gene (TaLr35PR2) were analyzed using bioinformatics methods. In the meanwhile, the gene expression profiles of TaLr35PR2 induced by P. triticina and chemical molecule at different time points were analyzed by semi-quantitative RT-PCR method combining genetools and SPSS software. In addition, the similar analysis was also conducted on the different tissues of wheat including roots, stems and leaves, and different growth stages of wheat.【Result】TaLr35PR2 coded an alkaline protein positioned in the vacuole contained a signal peptide. The protein sequence of TaLr35PR2 had high homology with other plants, and had the closest relationship with pathogenesis-related protein 2 gene in Triticum aestivum, but farthest relationship with the PR protein in rice. The expression profile analysis of TaLr35PR2 at the adult stage in wheat at different time points after P. triticina inoculation indicated that the transcription level changed little in the compatible interaction (Thatcher), but changed much in the incompatible interaction (TcLr35). The transcription level was much earlier and higher in the incompatible interaction than that in the compatible interaction at different points. Different tissues of the adult wheat TcLr35 detection results showed that the gene expression in leaves increased significantly with the extension of inoculation time, the expression level reached a peak at 48 hpi, but the gene was not expressed in roots at different time points, and there was a little expression level at 48 hpi in stems and changed a little at the later period, so its expression abundance was leaf＞stem＞root. In different periods of wheat growth and development, the TaLr35PR2 expressed at certain levels without inoculation both in TcLr35 and Thatcher except at one-leaf stage, and the expression levels were much higher both in the incompatible and compatible interactions than that in the Mock reaction, then expressed stably at the adult stage. In addition, the expression level of the TaLr35PR2 increased significantly after pre-treatments with chemical molecule such as salicylic acid (SA) and abscisic acid (ABA).【Conclusion】The expression of TaLr35PR2 was induced by P. triticina. TaLr35PR2 may be involved in the defense response against rust pathogen and participate in SA, ABA mediated signaling pathway and take part in defense reaction to P. triticina of wheat TcLr35 adult stage.

Screening of Putative Proteins in Vector Laodelphax striatellus Which are Interacted with Disease-Specific Protein of Rice stripe virus by Yeast Two-Hybrid Based on the Split-Ubiquitin

QIN Fa-Liang, LIU Wen-Wen, LI Li, WANG Xi-Feng

Scientia Agricultura Sinica. 2014, 47(14): 2784-2794. doi:10.3864/j.issn.0578-1752.2014.14.009

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【Objective】The objective of this study is to screen the proteins in the small brown planthopper (Laodelphax striatellus, SBPH) interacted with the disease-specific protein (SP) of Rice stripe virus and to further clarify their functions for understanding the mechanism of interactions between virus and vector insect.【Method】The high-quality total RNA of the SBPH was isolated and purified by TaKaRa D9086 OligtexTM-dT30 mRNA Purification Kit and used as the template to synthesize the double-strand cDNAs by SMART technology. The ds cDNAs dealed with SfiⅠ were ligated with vector pPR3-N for construction of a cDNA library with high quality. cDNA library was amplified by electro transformation and tested the quality and titer. The target gene (SP) was got by the primers designed with SfiⅠsequence. The gene sequence, SP, was ligated with vector pDHB1 and the product was called bait vector, pDHB1-SP. Then pDHB1-SP was transformed into the NMY51 yeast, and total proteins were extracted. Western Blot was applied to check whether SP was expressed or not and functional assay was used to identify whether the bait vector, pDHB1-SP, was suited to this yeast two-hybrid system. The library screening stringency was optimized by using a pilot screen. Proteins interacted with the bait pDHB1-SP were screened from the cDNA library using the yeast two-hybrid system based on the split-ubiquitin and analyzed using gene ontology (GO), then 20 proteins were screened for further interaction assay.【Result】The high quality RNA was extracted for synthetised ds cDNA by SMART technology and the compounded ds cDNA size was ranged from 0.1 to 4.5 kb. The unamplified cDNA library was consisted of 1.2×106 independed clones, the titer of library was 1.12×108 cfu/mL, and the average size of inserts was above 1.5 kb in the cDNA library. The result of restriction enzyme digestion suggested that the bait vector, pDHB1-SP, was constructed successfully. pDHB1-SP was expressed in NMY51 using Western Blot and suited to the yeast two-hybrid system by functional assay. A total of 534 clones from the library were got, and 76 proteins may be interacted with RSV SP after sequencing and blast. Twenty of them selected based on the subcellular localization of SP in SBPH and functional analysis in terms of GO were further confirmed by the β-galactosidase assay and co-transformation assay and the results suggested that they were all interacted with SP.【Conclusion】Some proteins interacted with RSV-SP were screened from the cDNA library of SBPH and are important for understanding the mechanism of interaction between RSV and vector insect.

Changes of Different Carbon Fractions Caused by Long-Term N Fertilization in Dryland Soil of the Loess Plateau

LI Xiao-Han-1, 2 , LI Fu-Cui-1, LIU Jin-Shan-1, 2 , HAO Ming-De-3, WANG Chao-Hui-1, 2

Scientia Agricultura Sinica. 2014, 47(14): 2795-2803. doi:10.3864/j.issn.0578-1752.2014.14.010

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【Objective】 Increased organic carbon (C) sequestration, especially the accumulation of organic C in soil, and decreased carbon losses from soil are of great importance for improving the dryland soil fertility and mitigating the greenhouse effect. Soils in the Loess Plateau areas are known typically lower in contents of organic C, and increasing application of nitrogen (N) fertilizer is proved to be the important tactical measure to increase crop yields, but information about how the N fertilizer input can affect the soil C is still lacking. 【Method】 A 23 years long-term experiment was conducted with winter wheat as test crop growing at five N rates of 0, 45, 90, 135, 180 kg N•hm-2 per year on a basal fertilization of 39 kg P2O5•hm-2 per year in dryland area of Loess Plateau. Soil samples were collected from 0 to 40 cm soil layers for each plots at harvest of winter wheat, in order to study the effects of long-term application of different nitrogen fertilizer rates on soil total carbon (TC), total organic carbon (TOC), light fraction organic carbon (LFOC) and inorganic carbon (IC), estimate the changes of TOC, LFOC and IC accumulation, and quantify the effects of N fertilizer application rates on different kinds of C in dryland soil. 【Result】 Obtained results showed that, with the increase of N fertilizer rates, the TC showed no significant change, while the TOC contents in 0 to 30 cm soil layers were increased by 7% to 28%, and the LFOC contents in 0-40 cm soil were increased by 31% to 106 %, but over application of N fertilizer showed no advantages on the organic carbon accumulation. Analysis of regression with the accumulation of different soil C fractions to N fertilizer application rates showed that, TOC accumulation in 0 to 30 cm soil layers reached the maximum of 36.6 Mg at 120 kg N•hm-2 and LFOC in 0 to 40 soil layers reached the maximum of 2.69 Mg at 161 kg N•hm-2, with the application of 1.0 kg fertilizer N per year led to an increase of TOC by 1.34 kg•hm-2 and LFOC by 0.31 kg•hm-2. In addition, the ratio of LFOC to TOC in 0 to 20 cm soil layers was found also increased with the increase of N fertilizer rate. In contrast, the IC content decreased significantly with the increase of N fertilizer application rates, especially in the 5 to 20 cm soil layers, where accumulation of IC was found decreased by 2.8 Mg at 180 kg N•hm-2, with the application of 1.0 kg fertilizer N per year led to a decrease of IC by 0.67 kg•hm-2. 【Conclusion】 Long term application of different rates of N fertilizer showed no significant effects on the amount of TC, but the composition of different C fraction was remarkably changed in the dryland soil of the Loess Plateau, with the accumulation of TOC increased due to increase of LFOC sequestration in soil, but the IC accumulation decreased. Therefore, rational application of N fertilizer is not only the key measure to increase the crop yield, but is also of great importance to increase the organic carbon sequestration and then the soil fertility. However, the loss of inorganic carbon from soil caused by application of N fertilizer should not be overlooked, and sufficient attentions and concerns should be paid to the effects from the losses of soil IC on the sustainability of agriculture, ecology and environment in this area.

Effects of Nitrogen Supply Methods on Root Growth, Yield and Nitrogen Use of Maize

QI Dong-Liang, WU Xue, HU Tian-Tian

Scientia Agricultura Sinica. 2014, 47(14): 2804-2813. doi:10.3864/j.issn.0578-1752.2014.14.011

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【Objective】Ridge-furrow irrigation has been promoted widely in northwestern China, but short in alternative nitrogen application. This article described the effects of different nitrogen application methods on root growth, yield and nitrogen use efficiency of spring maize under field condition in conventional furrow irrigation situation, in order to reveal the pattern of root growth and distribution, yield, and nitrogen use under different nitrogen application methods and explore the reasonable nitrogen applications fitting to arid and semi-arid region in northwestern China.【Method】Spring maize variety Jinsui 4 was used as the experimental materials and was planted with ridge-furrow irrigation for two consecutive years. There were three treatments: conventional nitrogen supply (CN, nitrogen was applied evenly on both furrows), alternate nitrogen supply (AN, nitrogen was applied alternately in one of the furrows) and fixed nitrogen supply (FN, nitrogen was applied fixed in one furrow). Nitrogen was applied at 200 kg N•hm-2 in each treatment. Urea nitrogen was selected and applied at three times: 50%, 25% and 25% with basal application in sowing, topdressing in spike formation and tasseling stages, respectively. Calcium superphosphate was applied as a base fertilizer, evenly spreaded at the rate of 45 kg P2O5•hm-2. The irrigation norm was 3 750 m3•hm-2 was conducted after sowing and at the stages of jointing, spike formation, tasseling and filling, respectively. Root lengths under the plant, in the right and left of the plant of maize in the soil layers of 0-100 cm were measured at the growth stages of maize tasselling, filling and maturity stages of maize, and each 20 cm as one layer. Root length density was calculated through the body of the sampling. Yield and the total nitrogen of the plant were measured after harvest and the nitrogen use efficiency was calculated. 【Result】The results showed that, maize roots were mainly gathered under the plant in the soil layer of 0-40 cm. Root length density showed a decreasing trend with the soil depth increasing. In general, the root length density of maize was the largest in CN, the second place was in AN and smallest in FN during the monitoring period. Root length density was larger in CN in the soil layer of 0-40 cm while was larger in FN in the soil layer 60-100 cm. As in FN, root length density had a significant difference between right and left sides of the plant. The root length densities were nearly the same between right and left side of the plant in CN. The average nitrogen uptake rate, yield and nitrogen use efficiency in CN in two years was 217.8 kg N•hm-2, 10 318 kg•hm-2 and 47.3 kg•kg-1N, respectively. Compared with FN and AN, the nitrogen uptake rate in CN improved averagely by 4.28% and 2.50%, respectively; the yield improved averagely by 8.45% and 4.42%, respectively; the nitrogen use efficiency improved averagely by 4.08% and 1.87%, respectively. 【Conclusion】 Obviously, CN could maintain a larger root mass and had a most uniform distribution of roots between right and left sides of the plant during the monitoring period. Roots distribution between right and left sides of the plant had a significant difference under FN. The AN was located between CN and FN in maintaining root growth. As maintaining of the root growth and yield formation in three treatments, the CN was the best, AN was the second and FN was the worst. It was suggested that in condition of conventional furrow irrigation, nitrogen fertilizer should be applied evenly on both furrows and it is a good way for nitrogen applications.

Construction of a Genetic Linkage Map and QTL Analysis of Fruit-Associated Traits in Watermelon

LIU Chuan-Qi, GAO Peng, LUAN Fei-Shi

Scientia Agricultura Sinica. 2014, 47(14): 2814-2829. doi:10.3864/j.issn.0578-1752.2014.14.012

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【Objective】The purpose of this study is to construct a molecular genetic map and map the QTL of the fruit-associated traits with CAPS and SSR markers in watermelon, which will provide a theoretical basis for traits improving, gene fine mapping and gene cloning.【Method】Fruits of female parent PI186490, male parent LSW-177 and F2 population derived from the cross between the two watermelon strains were picked in 40 days after pollination. Fruit shape index, center and edge brix, center and edge flesh firmness, rind hardness, seed length, seed width, seed thickness and 100-seed-weight were investigated correspondingly, then the obtained data were analyzed by SPSS19. Both parent materials genomes were resequenced by Illumina HiSeq 2000 platform for high-throughput sequencing, outputed 10G each data sample, covered more than 20× of watermelon genome. With the published genome as a reference, the obtained data were assembled with bwa, and explored for the SNP by Samtools. The sequence 1 000 bp around the SNP loci was extracted by self perl script and then inputed into SNP2CAPS to transform into CAPS markers. Twenty CAPS restriction sites were selected evenly on the 11 chromosomes. CAPS primers were designed 100-500bp around the mutation by Primer Premier 5 for PCR amplification and digestion detection. 1% agarose gel electrophoresis was used to detect the digestion products. SSR markers in this experiment were come from the published literature. The products of SSR-PCR were detected by polyacrylamid gel electrophoresis. All the molecular data were tested by chi-square. Markers which were confirmed the proportion 1﹕2﹕1 were chosen for the genetic linkage map. The genetic linkage map was constructed by Mapmaker/Exp version 3.0. The markers were grouped with the order ‘Group’. The number of the markers in the group which was less than 8 was sequenced faultlessly with the order ‘Compare’, which was more than 8 was ordered with the order ‘Try’. Map Chart 2.1 was used for drawing this genetic linkage map. QTL Network 2.0 was used for QTL analysis. 1 000 times repeats were done with the replacement testing, the critical threshold was P=0.005, and the method of constructing the map was composite interval mapping. The whole genome was scanned on every chromosome with 1 cM walking speed. QTL additive effect and epistatic effect were analyzed by the software.【Result】This genetic linkage map contained 16 linkage groups and included 87 CAPS markers and SSR markers. The map was 1 484.3 cM and the average distance between two makers was 15.46 cM. Mapping the QTL of the fruit-associated traits and analyzed by software QTL Network 2.0, and a total of 8 additive QTL and one pair of epistatic QTL were detected. Among the additive loci, 1 is for fruit shape index(QFSI 1), 1 for center brix (QCBR), 1 for center flesh firmness(QCFF), 1 for edge flesh firmness(QEFF), 1 for seed length(QSL), and 3 for seed width(QSWD 1, QSWD 2, QSWD 3). The epistatic loci, FSI 2 and FSI 3 are for fruit shape index. Phenotypic contribution rate of 10% or more have six QTL, which explained 11.7% -18.8% of the genetic variation. All of the QTL explained 7.12%-18.8% of the phenotypic variation.【Conclusion】A molecular genetic linkage map composed mainly of CAPS markers was constructed. Eight additive QTL and one pair of epistatic QTL, which control the fruit-associated traits of watermelon were located, thus providing a scientific basis for further fine positioning and cloning the genes controlling superior traits of fruit of watermelon.

The Influence of Different Crops Rotation on the Environment of Soil and Physiological Characteristics of Malus hupehensis Rehd. Seedlings

吕Yi-1 , SONG Fu-Hai-1, LI Yuan-Yuan-2, SHEN Xiang-1, CHEN Xue-Sen-1, WU Shu-Jing-1, MAO Zhi-Quan-1

Scientia Agricultura Sinica. 2014, 47(14): 2830-2839. doi:10.3864/j.issn.0578-1752.2014.14.013

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【Objective】Wit the soils of 25 years continuous cropping apple orchard as the object, the influence of different crops rotation on soil environment of continuous cropping apple orchard and physiological indexes of Malus hupehensis Rehd. seedling was studied, then a preferable rotation planting crop was chosen, in order to provide a gist for the prevention and control of apple continuous cropping obstacles.【Method】 Wheat, peanut, alfalfa, shallot, and potato were planted respectively in the soil, which was collected from the old apple orchard and blended evenly, then the soil organic matter, content of available N, P, K, effective Fe , Mn, Cu and Zn, the change of the number of soil bacteria, fungi and actinomycetes, and the change of contents of soil phenolic acids were determined. At the same time, potting M. hupehensis Rehd. seedlings after rotation of different crops. The biomass and root activity, SOD, POD, and CAT enzyme activities of M. hupehensis Rehd. seedlings were determined. 【Result】The content of soil organic matter increased significantly in the crop rotation soil of wheat and shallot when compared with apple continuous cropping soil, and the increments were 48.37% and 36.7%, respectively. Soil organic matter content of potatoes crop rotation and fallowing was reduced by 8.61% and 9.26%, respectively, compared with CK, and potato rotation significantly enhanced the content of available N, P, effective Cu, Fe, and Mn, compared with controls and increased by 217.32%, 217.32%, 240.55%, 91.38% and 158.30%, respectively. Peanut rotation remarkably reduced the content of available potassium, but increased the contents of effective Mn and Fe. Simultaneously rotation with wheat, peanut and shallot increased the number of soil bacteria, inhibited the breeding harmful fungus in the soil. Among them, bacteria amounts of shallot rotation increased by 200%, fungi amount decreased by 44.7%, bacteria amounts of shallot rotation increased by 141.9%, shallot rotation and Wheat rotation enhanced the ratio of bacteria and fungi by 435.8% and 211.1%, respectively. Shallot rotation markedly reduced the content of phenolic acids in the soil by 45.3%, among them, the content of phloridzin decreased by 84.2%. Compared with potato rotation, the content of phenolic acids in shallot rotation decreased by 241.15%, and compared with wheat rotation decreased by 190.07%. Compared with CK, Shallot rotation significantly improved the root SOD, POD, and CAT enzyme activities of M. hupehensis Rehd., and increased by 48.12%, 58.33% and 65.77%, respectively. It could be seen that Shallot rotation significantly increased the ground fresh weight of M. hupehensis Rehd. by 49.7%, underground fresh weight increased by 76.5% compared with CK. Shallot rotation significantly increased the ground fresh weight of M. hupehensis Rehd. by 40.63% compared with peanut rotation, and underground fresh weight increased by 56.84% compared with wheat rotation.【Conclusion】There exist larger differences in the effect of continuous cropping soil environment and root of M. hupehensis Rehd. when rotation cropping with different plants. Shallot rotation can increase the content of soil organic matter, enhance the ratio of bacteria and fungi, reduce the content of phenolic acids in the soil, improve the root enzyme activity of M. hupehensis Rehd., and increase the ground and underground biomass. From the perspective of prevention and control of apple continuous cropping obstacles, rotation shallot is more advantageous to reduce the apple continuous cropping obstacles.

Comparison of the Constitutions, Distribution, and Antioxidant Activities of Polyphenols from Different Varieties of Tartary Buckwheat Seed Produced from Different Regions of China

LIU Qin, ZHANG Wei-Na, ZHU Yuan-Yuan, HU Qiu-Hui

Scientia Agricultura Sinica. 2014, 47(14): 2840-2852. doi:10.3864/j.issn.0578-1752.2014.14.014

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【Objective】 The constitution, content and distribution of polyphenols in hull, bran, flour and whole grain of different varieties of tartary buckwheats from different regions of China were studied. The antioxidant activities of polyphenol extracts from different parts and samples of tartary buckwheat were compared. The aim of the study is to obtain the scientific data for improvement of tartary buckwheat breeding and further processing. 【Method】 Total phenolic contents (TPCs) of hull, bran, flour and whole grain of 17 varieties of tartary buckwheat were firstly compared by Folin–Ciocalteu reagent method. The polyphenol constitutions and individual phenolics content were analyzed by HPLC coupled with mass spectrometry (HPLC/MS) and tandem MS (MS/MS). The antioxidant activities of different polyphenol extracts were compared by DPPH and ORAC methods. 【Result】 Both Folin–Ciocalteu reagent and HPLC methods showed that tartary buckwheat bran had the highest TPC. Based on HPLC results, the average TPC in bran of all samples was 4 410.23 mg/100 g, which was 4.8 times of average TPC in hull and 15.5 times of average TPC in buckwheat flour. There were eight polyphenol species identified by HPLC/MS and MS/MS, among them Rutin was the major species. The contents of rutin in hull, bran and flour were 342.55-1 758.06 mg/100 g, 2 653.84-5 488.55 and 148.23-542.68 mg/100 g, which accounted for 82.13%-90.16%, 87.71%-92.09% and 80.85%-86.53% of TPC in hull, bran, and flour, respectively. Chlorogenic acid was only detected in hull samples. While, hyperoside mainly occur in hull and bran, and sinapic acid was firstly detected in tartary buckwheat at very low concentration. Polyphenol distribution and contents in buckwheats from different regions were significantly different. The TPCs in hulls from Yunnan were significantly lower than those from Shaanxi and Sichuan. However, the average TPC in brans of buckwheats from Yunnan was higher than those from Sichuan, and closed to those from Shaanxi. The whole grains from Yunnan had the highest TPC and antioxidant activities. The DPPH and ORAC results showed that for all samples, brans had the highest antioxidant activities, while flours had the lowest antioxidant activities. Overall, the buckwheat samples from Shaanxi had the highest DPPH and ORCA values no matter for hull, bran and flour. The hulls and flours from Yunnan had the lowest value in both DPPH and ORAC values, but had the highest antioxidant capability for whole grains. 【Conclusion】HPLC coupled with mass spectrometry is very powerful in studying the polyphenol in plants. Results of this study showed that the polyphenol distribution and contents in tartary buckwheat from different regions of China were significantly different. And the TPCs positively correlated with the antioxidant activities.

Genetic Diversity and Population Structure of Chinese and American Alfalfa（Medicago Sativa. L） Germplasm Assessed by SSR Markers

QIANG Hai-Ping-1, YU Guo-Hui-1, LIU Hai-Quan-1, GAO Hong-Wen-1, LIU Gui-Bo-2, ZHAO Hai-Ming-2, WANG Zan-1

Scientia Agricultura Sinica. 2014, 47(14): 2853-2862. doi:10.3864/j.issn.0578-1752.2014.14.015

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【Objective】Alfalfa is the most important cultivated forage across the world. However, for little improvement in forage yield and quality in recent years, conventional breeding methods are far from satisfying the practical need. On the one hand, the improvement in cultivars relies on the quantity of breeding resources, on the other hand, it relies on the understanding of the genetic basis of agronomic traits. Based on SSR markers, the study was conducted to analyze the genetic diversity and population structure of the germplasm from China and the United States and provide basic information on mining beneficial markers and alleles significantly associated with important quantitative and quality traits of alfalfa when using genome-wide association study to facilitate breeding process.【Method】In total, 40 SSR loci, covered the entire alfalfa genome with 3 to 9 SSR loci chosen on each chromosome, were used to conduct the genomic scanning of a collection of 100 Chinese and American alfalfa genotypes out of 16 main cultivars and to determine the genetic diversity and population structure. For Chinese cultivars, 8 genotypes were chosen for each cultivar; while for American cultivars, 4 genotypes per cultivar. Based on the admixture model, the software program STRUCTURE was used to determine the population structure of alfalfa. The optimal value of K (the number of clusters) was deducted by evaluating K=1-6. Length of burn-in of the Markov Chain Monte Carlo (MCMC) iterations was set to 10 000 and data were collected over 100 000 MCMC iterations in each run. Ten iterations per K were conducted. The optimal value of K was identified using two methods. The result was displayed by Structure Harvester and Distruct. Further, the principle component analysis and clustering analysis were conducted to justify the result of population structure. Based on the result of population structure, the genetic diversity of all samples and two populations were analyzed.【Result】A total of 446 alleles were detected using 40 SSR markers while 11.2 alleles was detected on average per locus with the range of 3-27, expected heterozygosity was 0.742 on average with the range of 0.493-0.901 and polymorphism information content (PIC) was 0.707 with the range of 0.493-0.901. These parameters indicated high genetic information in Chinese and American alfalfa cultivar resources. For the high genetic diversity displayed by mtic238, mtic188, bf111, afctt1, bf641851, maa660456, aw361, etc., they were suitable to disclose the genetic diversity in Chinese and American alfalfa. In terms of different chromosomes, the second and eighth chromosomes possessed relatively high genetic diversity while the first relatively low. In the analysis of population structure based on the admixture model, two different methods determined the optimal K was 2, which meant 100 genotypes from 16 cultivars could be grouped into two sub-populations even with certain discrepancy. The principle component analysis and clustering analysis were consistent with the analysis of population structure. The genetic diversity of Chinese cultivars was slightly higher than American’s, but not significant. 【Conclusion】 The germplasms from China and the United States were relatively highly diversified and variable in genetic analyses, revealing high genetic diversity. Difference exists in the diversity of genotypes of both countries: Chinese germplasm is slightly higher than those from America. The fact that the population structure of all genotypes was not strictly consistent with the original countries is credited to the allogamy and wide genetic exchange in alfalfa.

Identification of the ORF2 and ORF3 of Porcine Circovirus Type 2-Like Virus P1 on the Protein and Transcriptional Levels

WANG Feng-Zhi-1, 2 , WEN Li-Bin-1, 2 , YAO Huo-Chun-1, HE Kong-Wang-2

Scientia Agricultura Sinica. 2014, 47(14): 2863-2871. doi:10.3864/j.issn.0578-1752.2014.14.016

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【Objective】 The objective of this study is to determine the presence of the open reading frames ORF2 and ORF3 in porcine circovirus type 2-like virus P1.【Method】 After transfection of P1 molecular cloning, extracted and purified the total RNA, then RT-PCR was developed for detection of the RNA with specific primers ORF2 and ORF3, respectively. The products of PCR were recovered using AxyprepTM DNA Gel Extraction Kit, and then Trans5α competetent cells were transformed, the single colony were selected and sequenced after tested positively by PCR. Simultaneously, the 5′- and 3′-ends of P1 ORF2 and ORF3 were amplified using RACE. Furthermore, B-cell epitopes of P1 ORF2 and ORF3 were predicted according to their sequences, and then the corresponding epitope peptides were synthesized using standard stepwise solid-phase synthesis method and coupled to keyhole limpet hemocyanin (KLH) to increase antigenicity. New Zealand White Rabbits were immunized with KLH conjugated peptides to prepare anti-P1 ORF2 and -ORF3 polyclonal antibodies. Conventional indirect ELISA was applied to detect the titer, coated with the synthetic peptide antigen, and then their absorbency was measured under 450 nm wavelength. The highest serum dilution of S/N ≥ 2.1 was identified as the antibody titer. Reactivity of P1 and the two polyclonal antibodies were detected by immunohistochemistry, and the copies of P1 in transfected cells were determined by real-time PCR, simultaneously. 【Result】 The RT-PCR with the specific primers of ORF2 and ORF3, could amplify the 111bp and 128bp target fragments, which had 100% sequence homology with P1 ORF2 and ORF3, respectively. The 5′- and 3′- RACE ends of P1 ORF2 located at sites 11（G） and 168（T), respectively; the 5′- and 3′- RACE ends of P1 ORF3 located at sites 285（T） and 579（A）, respectively. According to the prediction, the amino acid sequences of the synthesized epitope peptides were ORF2: CLSRLPQSERPPGRW and ORF3：CVYPKVRERRVLKMP. Titers of the polyclonal antibodies were both higher than 1:512000, which were prepared from New Zealand White Rabbits immunized with KLH conjugated peptides of ORF2 or ORF3. Besides, the polyclonal antibodies could develop specific chromogenic reaction with P1. Dyeing results of the Blue DAB chromogenic reagent kit were hyacinthine, mostly in cytoplasm and few in nucleus, while the control groups were no chromogenic reaction. The results of real-time PCR showed that P1 could propagate in the transfected cells, and 104 h after transfection harvested the largest proliferation.【Conclusion】 The presence of P1 ORF2 and ORF3 was determined at the transcriptional and protein levels by transcriptional analysis and immunohistochemistry.

Genome-Wide Association Study Reveals Candidate Susceptibility Loci for Pig Scrotal Hernia Using Both F2 Intercross and Outbred Populations

SU Ying-1, RUAN Guo-Rong-2, LONG Yi-1, YANG Bin-1, ZHANG Zhi-Yan-1, DENG Wei-Yun-1, WU Li-Hua-1, 吕Xian-Shan-1 , AI Hua-Shui-1, XIAO Shi-Jun-1, REN Jun-1, HUANG Lu-Sheng-1, DING Neng-Shui-1

Scientia Agricultura Sinica. 2014, 47(14): 2872-2880. doi:10.3864/j.issn.0578-1752.2014.14.017

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【Objective】Isolation of susceptible loci and positional candidate genes associated with pig scrotal hernia through case-control genome-wide association study (GWAS) based on haplotypes in a F2 White Duroc × Erhualian intercross and it’s replication in outbred trio families.【Method】Phenotypic data and Illumina porcine 60K SNP genotypes were collected from 1020 individuals in the White Duroc × Erhualian intercross (containing 19 affected pigs) and outbred trio families (containing 237 scrotal hernia pigs). Quality control was carried out using PLINK v1.07 for each population， separately. Samples of low (<90%) call rate were removed. SNP markers were removed if they had genotype-missing rates ＞0.1, or Hardy-Weinberg P≤10-3(based on Chi-squared test) or minor allele frequencies (MAF) ＜0.05, and SNPs on sex chromosomes or with unknown positions were also excluded. PHASEBOOK package was used to infer haplotypes from the genotyped individuals and to assign them to K ancestral haplotype clusters by hidden monto carlo markov models. The software GLASCOW based on generalized linear mixed models was used to carry out GWAS for pig scrotal hernia in the F2 intercross by the ancestral haplotype and significant SNPs were chosen to perform transmission disequilibrium test (TDT) based on both single SNP markers and haplotypes in the outbred population (containing 237 affected pigs) by Haploview. 【Result】After quality control, all samples in F2 population and 724 samples in outbred populations passed the filter and a final set of 38 033 SNPs and 96 SNPs were selected for GWAS and TDT analysis. In total, 108 SNPs with Bonferroni chromosome-wise significance (P＜2.63×10-5) were identified to be associated with scrotal hernia in F2 intercross, and located on SSC2, SSC8 and SSC17, respectively. The most promising SNP was located on SSC17. Five significant SNPs, located in on nearby IQGAP2，CHMP4B，SERINC3，ZNF334 genes, were replicated (located on SSC2 and SSC17) in outbred trio families by TDT analysis. TDT analysis based on haplotype revealed that two haplotypes contribute to risk of scrotal hernia, one of which was overlapped with two significant SNP on SSC17 in the TDT analysis based on single marker. IQGAP2 and CHMP4B genes close to the SNPs most associated with pig scrotal hernia were selected as positional candidate genes according to its biological and physiological functions.【Conclusion】A GWAS with small samples was conducted and variation and replication test was performed in a larger populations, which revealed 5 susceptible loci for pig scrotal hernia both in the F2 intercross and the outbred populations. IQGAP2 and CHMP4B may play a role in the risk of scrotal hernia, and deeper analysis should be carried out in the future study.

Molecular Mapping and Physiological Characterization of a Novel Mutant rl15(t) in Rice

ZHANG Li-Xia, LIU He-Qin, YU Xin, WANG Lin-You, FAN Hong-Huan, JIN Qing-Sheng, WANG Jian-Jun

Scientia Agricultura Sinica. 2014, 47(14): 2881-2888. doi:10.3864/j.issn.0578-1752.2014.14.018

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【Objective】Phenotypic and physiological characteristics of an environmentally induced rolled leaf mutant were studied in present paper. Meanwhile, the mutant gene was preliminarily mapped on rice chromosome. 【Method】A rolled leaf mutant, named rl15 (t) (rolled leaf 15), was obtained by 60Coγ-ray mutagenesis from japonica cv. Nipponbare. By field identification, the phenotypes and main agronomic traits of the mutant were investigated. Different temperature and relative humidity treatments could reveal the environment factors that affect leaves rolling in mutant. Both of rl15(t) and wild type at heading stage were divided into 6 groups and were treated with temperature at 24℃, 29℃ and 34℃, and relative humidity at 60% and 95%. After treatment for 1.5 h, flag leaves were used to measure leaf rolling index (LRI). From 6:00 AM to 18:00 PM, the photosynthesis rate (Pn), transpiration rate (Tr), stamatal conductance (Gs) of flag leaves in rl15(t) and wild type were measured at 2-hour intervals by using the portable gas exchange system Li-6400, meanwhile, leaf water potential was measured by WP4 dewpoint potential meter. These physiological characteristics were analyzed and compared between rl15(t) and wild type. The rl15(t) mutant was crossed with the wild type Nipponbare, leaf phenotype of the F1 progeny and segregation ratio of flat leaf plants and rolled leaf plants in F2 population were investigated. On the basis of BSA method according to Michelmore et al., preliminarily mapping of the candidate mutant gene were conducted using a F2 population derived from rl15(t) crossed with indica line Zhenshan97B.【Result】Compared with the wild type, the mutant plants have shortened plant height, reduced tiller numbers, shorter panicle, smaller grains, delayed heading duration, shorter and narrowed leaves. All of the leaves in rl15(t) were observed to highly inward roll at midday hours under sunny conditions, whereas were flat or slightly inward rolled under rainy conditions or at early morning and sunset under sunny conditions. Experiments of different temperature and relative humidity treatments showed that leaf rolling index in rl15(t) mutant were mainly depended on air humidity and could be promoted by high temperature. The photosynthesis rate, transpiration rate, stamatal conductance and leaf water potential of flag leaves in mutant were extremely lower than those in the wild type at midday hours under sunny conditions. However, the instantaneous water use efficiency (WUE) was similar to that of wild type at 6:00, 12:00, 18:00 o’clock, whereas in other time of a day, WUE was dramatically higher than that of the wild type. F1 plants derived from crossing rl15(t) with wild Nipponbare showed normal flat leaves, The segregation ratio of flat leaf plants to rolled leaf plants in F2 population was consistent with the inheritance of single recessive nuclear locus. Further molecular genetics studies revealed that RL15(t) was mapped on the long arm of rice chromosome 10 between SSR markers RM25302 and RM25343, with genetic distances of 0.8 cM and 2.0 cM, respectively. 【Conclusion】Mutant rl15 (t) was environmentally induced rolled leaf in phenotype. RL15(t) gene is located between SSR markers RM25302 and RM25343. In these distance segment, it hasn’t any similar phenotype genes reported up to now. So, RL15(t) gene would be a putative novel rolled leaf gene.

Isolation of a Maize Wound-Induced Gene Promoter and Characterization of the Gene Expression in Maize

LIAN Yun-1, 2 , LIU Yun-Jun-1, WANG Guo-Ying-1

Scientia Agricultura Sinica. 2014, 47(14): 2889-2896. doi:10.3864/j.issn.0578-1752.2014.14.019

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【Objective】 Protease inhibitor is an important defense protein in plant for resistance to infection or feeding by pathogen and animals. Maize Wip1(wound-induced protein) is the member of Bowman-Birk inhibitor(BBI) gene family. To provide new information for the application of resistance gene to insect in plant, this experiment was designated to understand the activity of Wip1 promoter, assay spatial and temporal expression of Wip1 gene and its response to phytohormones and abiotic stresses.【Method】A clone containing the upstream region of Wip1 was isolated and sequenced from maize leaf cDNA. The promoter sequences of Wip1 gene was inserted into binary expression vector p1300 to produce recombinant plasmids p1300-Wip1-GUS, which was transferred into Agrobacterium LBA4404 by freeze thawing method. Functional analysis of the promoter was conducted through heterologous expression in maize callus by agro-infiltration method. Total RNA was isolated from maize tissues using hot phenol method. Purified RNA was subjected to formaldehyde-containing-agarose gel, blotted onto a nylon membrane, and Northern blotting was performed with [α-32P] dCTP-labeled probe to characterized tissue-speci?c location, phytohormones and abiotic stresses analysis of Wip1. 【Result】 Promoter region was 1 737 bp and coding sequence was 512 bp. The result showed that GUS activity driven by the Wip1 promoter was detected in maize callus tissues after infection by Agrobacterium haboring recombinant plasmids p1300-Wip1-GUS. The expression of Wip1 was assayed at various developmental stages. Northern blotting showed that Wip1 gene abundantly expressed in injured coleoptile, root, stem, leaf, ear, tassel, silk and husk, while expressed little in uninjured coleoptile and not expressed in other uninjured detection tissues. The results also showed that Wip1 was not affected by submergence, cold, dehydration, PEG, ABA, NaCl and IAA treatments. The dynamic expression of Wip1 gene under injure stress from 0 h to 24 h with 8 time points, was analyzed in maize leaf tissue. Wip1 gene was detected after stressing for 2 h, the expression level increased rapidly, and with a continually increasing trend from 4 h to 24 h. 【Conclusion】 Wip1 gene is induced by injure-specific stress. Wip1 promoter is an effective injure-specific promoter to express the desired genes in plants.

Separation and Purification of Polyphenol in Litchi Pulp by Macroporous Resin

SU Dong-Xiao-1, 2 , ZHANG Rui-Fen-1, ZHANG Ming-Wei-1, HUANG Fei-1, 2 , WEI Zhen-Cheng-1, ZHANG Yan-1, TI Hui-Hui-1, DENG Yuan-Yuan-1, TANG Xiao-Jun-1

Scientia Agricultura Sinica. 2014, 47(14): 2897-2906. doi:10.3864/j.issn.0578-1752.2014.14.020

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【Objective】Litchi (Litchi chinenesis Sonn.) is an important subtropical to tropical fruit which contain a high amount of phenolics. Previous studies found that litchi exhibits excellent antioxidant, radioprotective and hepatoprotective activities. The aim of this study is to screen a resin which has a good adsorption and desorption performance of phenols of litchi, and optimize the separation process parameters, thus providing a theoretical basis for establishing polyphenols purification technology and identification of polyphenols construction of litchi. The separation and purification process of litchi pulp polyphenol was established by macroporous resin.【Method】The static adsorption and desorption performance of eleven different polarities macroporous resins (HPD-200A, HPD-400A, HPD-100, HPD-500, HPD-600, HPD-722, HPD-826, D4020, NKA-II, NKA-9 and AB-8) to total phenolics and total flavonoids of fresh litchi pulp chilled acetone/water extracts were compared to select suitable resin for purification of phenolic compounds. Kinetic curves of adsorption were studied. The dynamic adsorption and desorption process parameters, including sample concentration (3.2, 1.6, 0.8 and 0.4 mg•mL-1), flow rate (4.5, 3.0 and 1.5 BV/h), and eluent of ethanol concentration (95%, 80% and 60%), of macroporous resin of HPD-826 were optimized. The phenolic compound types and contents of litchi pulp were analyzed by HPLC with the retention time of standard including catechin, gallic acid, chlorogenic acid, vanillic acid, clove acid, caffeic acid, epicatechin, ferulic acid, 4-methylcatechol, coumarin, rutin, quercetin, resveratrol and quercetin-3-O- rutinose-7-O-rhamnose.【Result】The results showed that HPD-826 macroporous resin exhibited the best capability of adsorption and desorption of total phenolics and total flavonoids in litchi pulp. And the macroporous resin reached equilibrium within 4 h. The optimal separating process parameters were as follows: the concentration of litchi pulp extract and the sampling rate were 0.8 mg/mL and 3.0 BV/h, respectively, and the elution concentration and flow velocity were 95% ethanol and 3.0B V/h, respectively. The contents of phenolic compounds of litchi pulp were deduced by about 15% compared to before purification, but the phenolic profiles of litchi pulp were not changed after adsorption and desorption by HPD-826 macroporous resin. Nine phenolic compounds, 3,4 dihydroxybenzoic acid, catechin, vanillic acid, caffeic acid, syringic acid, epicatechin, quercetin 3-O-rutinoside-7-O-α-L- rhamnosidase, ferulic acid and rutin, were preliminary identified by HPLC. The major phenolic profiles were quercetin 3-O- rutinoside-7-O-α-L-rhamnosidase, rutin and epicatechin. The percentage contribution of the three compounds to the total phenolic content was 94.37%.【Conclusion】In conclusion, HPD-826 macroporous resin could be applied to purify total phenolics and total flavonoids in litchi pulp.

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