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16 December 2015, Volume 48 Issue 24

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Fine Mapping and Candidate Gene Analysis of qGT8, A Major QTL for Grain Thickness in Rice

GAO Fang-yuan, LUO Zheng-liang, REN Juan-sheng, WU Xian-ting, LU Xian-jun, SU Xiang-wen, Lü Jian-qun, REN Guang-jun

Scientia Agricultura Sinica. 2015, 48(24):  4859-4871.  doi:10.3864/j.issn.0578-1752.2015.24.001

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【Objective】 The paper is order to identify quantitative trait loci (QTLs) for grain length (GL), width (GW) and thickness(GT), and further to narrow down the genomic region containing the major QTL for GT and analyze the possible candidate genes, which would help us to understand the genetic basis underlying the formation of slender grains and facilitate the efforts to the marker-assisted improvement of yield in a cytoplasmic male-sterile (CMS) maintainer line Chuan106B (C-106B). 【Method】 In this study, C-106B, which has a long slender grain type and good grain quality, was crossed with Chuan345B (C-345B), another CMS maintainer line with thicker and wider grain, to produce F₂ population for a preliminary QTL analysis. The chromosome interval of QTLs for grain shape was identified by composite interval mapping (CIM) using QTL Catographer v2.5. Then, a BC₃F₂ population derived from the same cross was developed and those plants with thinner grain were further picked out for the fine mapping of the major QTL (qGT8) for GT. Sequencing analysis and quantitative real-time PCR for the candidate gene were also conducted. In addition, a pair of nearisogenic lines (NIL) carrying the C-106B (NIL-gt8^C-106B) and C-345B (NIL-GT8^C-345B) alleles at qGT8 locus were bred and applied to survey for yield and quality traits, respectively.【Result】Significant differences were observed for GL, GW and GT between C-106B and C-345B. Using the phenotype and genotype data of the F₂ population, two QTLs for GL, three for GW and three for GT were identified. Among these, the major QTL for GL, accounting for 68.23% of phenotypic variance was mapped to the region between RM21892 and RM3589 on chromosome 7. The allele from C-106B was responsible for increasing GL by 0.47 mm at this locus. Two major QTLs (qGW8 and qGT8) controlled GW and GT, each explaining up to 26.48% and 34.89% of the variation, respectively, were found in the same interval of RM6070-RM447 on chromosome 8. The C-345B alleles contributed to the increase in GW and GT at the two loci. Furthermore, using 1732 plants with recessive phenotype (thinner grain) from the BC₃F₂ population and 11 markers including in the region RM6070-RM447, qGT8 was delimitated to a region bounded by insertion/deletion (Indel) markers SG930 and SG950, with a physical interval of 11.2 kb, which contains only one predicted gene (LOC_os08g41940, OsSPL16). Comparison of the nucleotide sequencesof the C-106B and C-345B alleles of qGT8 uncovered seven nucleotide changes within the promoter region upstream of the ATG start codon and five polymorphisms in the coding region. One of these, a 2-bp insertion（c.1006_1007 ins CT）in exon 3 at miR156 target site in qGT8 of C-106B, result in a frameshift mutation, which is probably associated with the formation of a more slender grain. Expression analysis indicated that qGT8 was preferentially accumulated in developing panicles. The expression patterns of qGT8 at eight panicle developmental stages were similar between C-106B and C-345B, which gradually increased at early stages (panicles length ranging 1-8 cm), peaked at the 8 cm young panicle and then declined in panicles of 12 cm to 23 cm in length. However, the levels of transcript for qGT8 were different at every developing stage between two parents. Comparison of the two NILs showed that NIL-GT8^C-345B increased significantly in GW, GT, 1000-grain weight, grain yield per plant and the percentage of grain with chalkiness, while no significant differences were observed in other measured traits such as GL, transparency, plant height, number of panicles per plant, panicle length, number of filled grains per panicle, seed setting ratio and heading date. 【Conclusion】 A major QTL affecting grain length in C-106B was mapped to a region flanked by markers RM21892 and RM3589 on chromosome 7, while two major QTLs (qGW8 and qGT8) governing grain width and thickness, respectively, were found in the same interval of RM6070-RM447 on chromosome 8. Furthermore, qGT8 was fine mapped to a DNA region only covering GW8 gene and this result suggested that qGT8 may play a key regulator role in grain shape and yield traits. However, a coupling link of the increasing for grain weight and high chalkiness in NILs implied its counteracting effects on grain yield and quality at same locus.

Cloning and Function Analysis of Cotton Transcription Factor GhGT-2

LI Yue, LIU Xiao-dong, XIE Zong-ming, DONG Yong-mei, WU Dong-mei, CHEN Shou-yi

Scientia Agricultura Sinica. 2015, 48(24):  4872-4884.  doi:10.3864/j.issn.0578-1752.2015.24.002

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【Objective】Cotton (Gossypium hirsutum L.), the most important textile crop worldwide, often encounters abiotic stresses during its growth season and its productivity is significantly limited by adverse factors. Trihelix transcription factors are important proteins involved in response to abiotic stresses in plants. Understanding the molecular mechanisms of the Trihelix transcription factor genein cotton will lay the foundation for improving the stress tolerance of cotton by gene manipulation.【Method】We searched the cotton ESTs database using BLAST (the Basic Local Alignment Search Tool). One GT unigene, named GhGT-2, was obtained. Sequence analysis of GhGT-2 was performed to confirm that this gene is a member of the Trihelix GT-2 subfamily gene. The total RNA from the leaves of cotton was used as the template to design the degenerate primers based on expressed sequence tags. The full-length cDNA of GhGT-2 were cloned using the method of Rapid amplification of cDNA Ends (RACE) combined with reverse transcription-PCR (RT-PCR). Homologous analysis and multiple alignments were performed with MEGA 5. SMART online tools were used for protein sequence analysis. Seedlings were treated with conditions simulating drought, salinity, cold and abscisic acid (ABA) at the 15-day-old seedling stage in Xin luzao 26. After treatment for 0 , 1 , 3 , 6 , and 12 h, the seedlings were harvested respectively. In the meantime, cotton roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA) were harvested at different growth periods. The expression profiles of the GhGT-2 in various tissues and under cold, drought, salt and ABA treatments were investigated by quantitative real-time PCR (qRT-PCR) analysis. The GhGT-2【Result】The full-length cDNA sequence was obtained successfully from upland cotton, which was 1 579 bp in lenght, containing a 1 428 bp open reading frame that encoded a protein of 476 amino acids with a predicted molecular weight of 54.07 kD and an isoelectric point of 8.96. SMART analysis showed GhGT-2 contained two typical SANT motifs of GT-2 subfamily. Phylogenetic analysis showed that GhGT-2 belonged to the GT-2 subfamily of the Trihelix family and was most closely related to AtGTL1 and PtaGTL1. Real-time quantitative PCR (qRT-PCR) analysis revealed that GhGT-2 constitutively expresses in all tested tissues, such as roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA). The expression level of GhGT-2 was highestin leaves, and was very low in roots. The expression profiling revealed that GhGT-2 was induced by ABA, high salt and drought at 3, 6, and 12 h points, but was weakly repressed at 1 h point. In contrast, expression of GhGT-2 was weakly repressed at 3, 6, and 12 h points under cold treatment apart from the 3 h point. These results suggested that GhGT-2 might be involved in stress resistance-related response of ABA signaling pathways in cotton plant. Using the Arabidopsis protoplasts assay system, we found that GhGT-2 protein was located in cell nuclei but its trans-activation activity could not be detected clearly. Electrophoretic Mobility Shift Assay (EMSA) showed that GhGT-2 protein could bind to GT2-Box, GT3-Box, GT-1b (BoxⅡ), MBS1, MRE1, MRE3 and MRE4. 【Conclusion】A full-length cDNA of GhGT-2 was isolated and the typical SNAT binding domain were found in the deduced protein, belonging to the Trihelix GT-2 subfamily. The expression of GhGT-2 was up-regulated under drought, high salt and ABA treatments, showing that GhGT-2 was involved in the ABA dependent stress-responding gene network. It was deduced that GhGT-2 may play important roles in upland cotton plant abiotic stress adaption. coding sequence was cloned onto GFP expressing vector pBI221, and GAL4 DNA-binding domain vector, transformed into Arabidopsis protoplasts to verify the localization in the cell and the transcriptional activation ability of the GhGT-2 protein. The gel-shift assay was performed to detect DNA binding elements.

Crossability Between Brassica napus with Hexaploid AⁿAⁿCⁿCⁿC^oC^o and Sclerotinia Resistance in the Hybrids

LIU Yao, DING Yi-juan,WANG Lei,WAN Hua-fang,MEI Jia-qin, QIAN Wei

Scientia Agricultura Sinica. 2015, 48(24):  4885-4891.  doi:10.3864/j.issn.0578-1752.2015.24.003

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【Objective】 It was previously proposed to transfer genetic components from Brassica oleracea into B. napus via the bridge of hexaploid derived from B. napus and B. oleracea. The present study was carried out to assess the potential of the hexaploid strategy in rapeseed improvement by investigating crossability between B. napus and hexaploid and evaluating fertility and sclerotinia resistance in the hybrids.【Method】 The hexaploid AⁿAⁿCⁿCⁿC^oC^o was developed by embryo rescue and chromosome doubling from a interspecific cross between B. napus var. Zhongshuang 9 and a wild B. oleracea accession with strong sclerotinia resistance. The crossability between the hexaploid and nine B. napus accessions was studied by investigating the seed setting rate of pollinated flowers, and the seed set from open- and self-pollination of the nine F₁ hybrids were checked to reveal the fertility of the F₁ generation. The sclerotinia resistance was assessed as well in the nine F₁ hybrids to investigate the improving efficiency of targeted trait. 【Result】 The hexaploid was identified to have 20 chromosomes from A genome and 36 chromosomes belonging to C genome, indicating the AⁿAⁿCⁿCⁿC^oC^o genome structure of the hexaploid. Genetic components from both Zhongshuang 9 and the wild B. oleracea were identified in the hexaploid. The seed set of hexaploid averaged 5.6 and 8.6 seeds/pod for self- and open- pollination, respectively. The pod setting rate of crosses between hexaploid and nine B. napus genotypes was 69.1%-92.9%, yielding 4.7-10.5 seeds per pod. A significant difference was detected among the nine crosses for both the pod setting rate and the seed set (P＜0.01). The F₁ hybrids showed normal pollen fertility but varied seed sets after selfing (11.4-20.7) and open pollination (19.9-26.1 seeds/pod) (P＜0.0001), with a positive correlation (r = 0.67*, P＜0.05) between the selfed- and open- seed sets. Based on stem resistance evaluation across 2013 and 2014, the relative susceptibility of hexaploid to Zhongshuang 9 was 0.47, while the relative susceptibility of the nine B. napus parental lines were 0.94-1.26, with significant differences among genotypes (P＜0.0001). The relative susceptibilities of F₁ hybrids were 0.56-1.10, being intermediate between corresponding parental lines and significantly associated with the resistance level of the B. napus parent (r = 0.78*, P＜0.05). Among the nine hybrids, five showed significantly lower relative susceptibilities as compared to their corresponding B. napus parents (P＜0.05), suggesting improved sclerotinia resistance in these F₁ hybrids. 【Conclusion】Although the crossability of B. napus with the AⁿAⁿCⁿCⁿC^oC^o hexaploid, which has good fertility, was significantly affected by parental B. napus, it is easy to develop F₁ hybrids with good fertility and markedly improved performances in the targeted traits.

TILLAGE & CULTIVATION·PHYSIOLOGY & ECOLOGY

Effects of Types of Controlled Released Nitrogen and Fertilization Modes on Yield and Dry Mass Production

XING Xiao-ming, LI Xiao-chun, DING Yan-feng, WANG Shao-hua, LIU Zheng-hui, TANG She, DING Cheng-qiang, LI Gang-hua, WEI Guang-bin

Scientia Agricultura Sinica. 2015, 48(24):  4892-4902.  doi:10.3864/j.issn.0578-1752.2015.24.004

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【Objective】This experiment seeks to investigate the effects of different types of controlled released nitrogen and fertilization modes on the yield, characteristics of photosynthesis and dry mass production. 【Method】 Local japonica rice of Wuyunjing 24 mechanical transplanting. Four N fertilizer types (urea, slow-release fertilizer blend, polymer-coated urea, sulfur-coated urea) and two fertilization modes, the base-tiller nitrogen fertilizer and single fertilization were conducted in Danyang, Jiangsu province, China, in 2013 and 2014, seven treatment including single fertilization of slow-release fertilizer blend(B-BSRB), single fertilization of polymer-coated urea(B-PCU), single fertilization of sulfur-coated urea(B-SCU), base-tiller nitrogen fertilizer of slow-release fertilizer blend(BT-BSRB), base-tiller nitrogen fertilizer of polymer-coated urea(BT-PCU), base-tiller nitrogen fertilizer of sulfur-coated urea(BT-SCU) and conventional split fertilization (CK). 【Result】 The yield of different types of controlled released nitrogen showed that, slow-release fertilizer blend >polymer-coated urea > sulfur-coated urea. The dry matter weight of the population, Leaf area index and photosynthetic potential of different controlled released nitrogens at the growth period showed that slow-release fertilizer blend > polymer-coated urea > sulfur-coated urea. Dry matter accumulation, its proportion of distribution and crop growth rate at the jointing stage showed slow-release fertilizer blend >polymer-coated urea > sulfur-coated urea, while at the maturity stage showed slow-release fertilizer blend> sulfur-coated urea> polymer- coated urea. All of them showed a trend of base-tiller nitrogen fertilizer treatment > single fertilization treatment. The yield of slow-releasing blending fertilizer with the base-tiller nitrogen fertilizer treatment (BT-BSRB) was 11.6 and 10.1 t hm^-2 in 2013 and 2014, respectively. Compared to conventional fertilization (CK), the yield of BT-BSRB increased by 9.4、12.2%, respectively.【Conclusion】Polymer-coated urea and sulfur-coated urea were a poor application on japonica rice, while slow-release fertilizer blend can significantly increase japonica rice yield and photosynthetic material production.

Effects of Spraying Uniconazole on Lodging Resistance of Culm and Yield in Common Buckwheat

LIU Xing-bei, WU Dong-qian, WANG Can, HU Dan, YANG Hao, SHE Heng-zhi, RUAN Ren-wu,YUAN Xiao-hui, YI Ze-lin

Scientia Agricultura Sinica. 2015, 48(24):  4903-4915.  doi:10.3864/j.issn.0578-1752.2015.24.005

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【Objective】Common buckwheat (Fagopyrum esculentum M.) has been praised as one of the green foods of human beings in the 21st century. Lodging is an important factor which limiting common buckwheat yield and quality worldwide, so exploring the effects of spraying uniconazole of different concentrations on culm could provide a theoretical basis for lodging resistance cultivation of common buckwheat. 【Method】 The field experiments were conducted as a randomized block design in 2013 and 2014 at the Xiema Experimental Station, Southwest University, China. Ningqiao 1, a moderate lodging resistance cultivar of common buckwheat, was used in this study. 0 mg·L^-1 (CK), 25 mg·L^-1 (T1), 50 mg·L^-1 (T2), 75 mg·L^-1 (T3), and 100 mg·L^-1 (T4) uniconazole were sprayed at the rate of 100 mL·m^-2 on common buckwheat leaf surfaces at the four-leaf stage. The culm snapping resistance, lodging index, culm morphology characteristics, and culm anatomical structure were measured and analyzed at the anthesis stage, filling stage, and maturity stage, respectively. The lodging behavior and yield were investigated at the maturity stage.【Result】The culm snapping resistance, culm wall thickness, and vascular bundle area increased and then decreased from the anthesis stage to maturity stage, and their maximum values appeared in the filling stage. The lodging index, plant height, culm gravity height, culm fresh weight, basal 2nd internode length, basal 2nd internode diameter, mechanical tissue thickness, and vascular bundle number increased from the anthesis stage to the maturity stage. The basal 2nd internode dry weight, filling degree, and mechanical tissue layer number increased from the anthesis stage to the filling stage, but then changes were not obvious. The yield, culm snapping resistance, basal 2nd internode diameter, basal 2nd internode dry weight, filling degree, mechanical tissue layer number, mechanical tissue thickness, culm wall thickness, vascular bundle number, and vascular bundle area increased with the increase of the concentration of uniconazole in the condition of CK-T3 treatments, while decreased with the increase of the concentration of uniconazole in the condition of T3-T4 treatments. The lodging percentage, lodging index, plant height, culm gravity height, culm fresh weight, and basal 2nd internode length decreased with the increase of the concentration of uniconazole in the condition of CK-T3 treatments, while it increased with the increase of the concentration of uniconazole in the condition of T3-T4 treatments. Compared with the control, the yield under the T1, T2, T3 and T4 treatments increased by 2.3%, 6.5%, 21.3%, and 11.3%, respectively, and the lodging percentage decreased by 17.9%, 40.7%, 84.0%, and 60.5%, respectively. There were significant differences in culm snapping resistance, lodging index, culm morphological structure, and culm anatomical structure among different treatments of uniconazole concentration. Plant height, culm gravity height, culm fresh weight, basal 2nd internode length , lodging index, lodging percentage were significant and negatively correlated with the yield. However, the basal 2nd internode diameter, basal 2nd internode dry weight, filling degree, mechanical tissue layer number, mechanical tissue thickness , culm wall thickness, vascular bundle area, vascular bundle number and culm snapping resistance are significantly and positively correlated with the yield. The culm snapping resistance, basal 2nd internode diameter, basal 2nd internode dry weight, filling degree, mechanical tissue layer number, mechanical tissue thickness, culm wall thickness, vascular bundle number, and vascular bundle area showed a trend of T3＞T4＞T2＞T1＞CK, while the lodging index, plant height, culm gravity height, culm fresh weight, and basal 2nd internode length showed a trend of CK＞T1＞T2＞T4＞T3.【Conclusion】In this research, when uniconazole was sprayed 75 mg·L^-1, the culm structure could be optimized effectively, the culm quality improved, the lodging risk decreased and the yield of common buckwheat was enhanced. The result would lay a foundation of lodging resistance cultivation of common buckwheat.

PLANT PROTECTION

Effect of Imazethapyr on Millet Jigu 33 Growth and Its Safety on Succeeding Crops

ZHANG Ting, SHI Zhi-gang, WANG Gen-ping, CHENG Ru-hong, CHEN Yuan, JI Xiao-mian, YANG Wei-hong

Scientia Agricultura Sinica. 2015, 48(24):  4916-4923.  doi:10.3864/j.issn.0578-1752.2015.24.006

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【Objective】 Jigu 33 is a new imazethapyr-resistant millet variety. Chemical seedling thinning and weed control can be realized by applying proper imazethapyr herbicides in large-scale cultivation and production. The objective of this study is to examine the effect of imazethapyr on the growth of millet Jigu 33 and its safety on succeeding crops, and to provide references for the application and promotion of Jigu 33.【Method】The experiment was carried out at the Qiema test station of the Institute of Millet Crops of Hebei Academy of Agricultural and Forestry Sciences with 6 dose levels of imazethapyr (0, 56.25, 75, 112.5, 150, 225 g a.i./hm²) from May 2013 to June 2015. The effect of imazethapyr on millet Jigu 33 growth was tested in the field. Jigu 33 seedling height and fresh weight were investigated on 7, 10, 13, 16, 20, 25 and 30 DAS (days after spraying); plant height, spike length, spike diameter and yield were investigated in the mature period; the residue of imazethapyr in millet, chaff, plant and soil were detected. Its safety on succeeding crops (Chinese cabbage, Jigu 19, Jigu 33, sugar beet, sorghum, maize, wheat and cotton) was tested on 30, 60 days and 1 year after sowing in net room.【Result】The growth of millet Jigu33 was normal at 56.25 to 225 g a.i./hm² and there was no evident injury. The effect on Jigu 33 seedling height and fresh weight in different periods after spraying showed that there was no obvious difference between the treatment and control. The sensitive period for seedling height was at the early jointing stage from 13 to 16 DAS, and the sensitive period for seedling weight was on the late jointing stage and the early jointing stage from 20 to 25 DAS. Then the inhibition was lightened gradually, and the growth of millet Jigu 33 was normal at harvest time, the yield of which was higher than the control. The residue was only detected in plant at 225 g a.i./hm² dose level with (0.01±0.006) mg?kg^-1, which was less than LOQ. The inhibition on the succeeding crops was also lightened gradually as time progressed. The plant height of the succeeding crops one year after sowing had significant differences. Of all the succeeding crops, Chinese cabbage and sorghum were sensitive with significant yield loss. In the spraying imazethapyr field in the summer millets region, the rotation cropping of Jigu 33 could be wheat, maize and cotton.【Conclusion】The growth and yield of millet Jigu 33 was normal at 56.25 to 225 g a.i./hm². The inhibition on the succeeding crops was lightened gradually as time progressed. The rotation cropping of Jigu 33 could be wheat, maize and cotton. In production, more attention should be paid to the control of the dosage and the rational rotation.

Phylogenetic Study of Colletotrichum Species Associated with Camellia sinensis from the Major Tea Areas in China

WANG Yu-chun, HAO Xin-yuan, HUANG Yu-ting, YUE Chuan, WANG Bo, CAO Hong-li, WANG Lu, WANG Xin-chao, YANG Ya-jun, XIAO Bin

Scientia Agricultura Sinica. 2015, 48(24):  4924-4935.  doi:10.3864/j.issn.0578-1752.2015.24.007

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【Objective】 Anthracnose pathogens can infect tea leaves, causing extensive necrosis of leaves and defoliation. The objective of this study is to identify the pathogens causing anthracnose on tea plants from the major tea areas in China, and to provide an important scientific basis for tea plant disease management in the future. 【Method】 Colletotrichum species associated with anthracnose diseases of tea plants were isolated using single spore isolation and confirmed by DNA sequencing of the partial sequences of β-tubulin and rDNA-ITS region. Neighbor-Joining analysis was performed to establish the multilocus phylogeny using MEGA 6.0. The morphology of isolated strains growing on fresh PDA plates was also described. Pathogenicity of typical isolates was further verified by inoculation assay on the tea varieties of ‘Longjing 43’ and ‘Zhongcha 108’ with and without wound treatments. 【Result】 A total of 78 Colletotrichum strains were isolated from tea plants in 15 provinces (municipality, autonomous region) of China. The 2-locus (ITS, TUB2) dataset for phylogenetic analysis results showed that isolates clustered in three clades, 45 isolates clustered with the ex-type isolate of C. camelliae, 28 isolates clustered with C. fructicola, 5 isolates clustered with C. siamense and all isolates belonged to the C. gloeosporioides species complex. Moreover, the results showed that C. camelliae was the most widely distributed species in the tea regions of China, reflecting that it was the dominant Colletotrichum species on Camellia sinensis. Meanwhile, morphological analysis showed that C. camelliae colonies on PDA growth rate 11.8 mm·d^-1, aerial mycelium white, dense, cottony; setae and sclerotium were not observed; conidiophores and conidiogenous cells were not observed; conidia hyaline, smooth-walled, cylindrical with obtuse ends, sometimes narrowed at the centre or towards the base, (8-15) µm×(3-6) µm; mycelial appressoria brown, clavate, irregularly shaped, branched, (8-10.5) µm×(6.5-8) µm; conidial appressoria were not observed. C. fructicola colonies on PDA growth rate 6.76 mm·d^-1, aerial mycelium white, reverse brown in the centre, white at the margin, dense, cottony; setae and sclerotium were not observed. Conidiophores hyaline, septate, branched; conidiogenous cells hyaline, cylindrical, (7-18) μm, apex (1-2) μm diam. Conidia hyaline, cylindrical, both ends rounded, sometimes narrowed at the centre, (10-15) µm× (3-3.5) µm; mycelial appressoria dark brown, cylindrical, (6.5-8) µm×(3.5-5.5) µm; conidial appressoria dark brown or brown, rounded, (5-7) µm×(5-6.5) µm. C. siamense colonies on PDA growth rate 7.6 mm·d^-1, white, dense, cottony; setae and sclerotium were not observed; Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical, 8-16 μm, apex 1-2 μm diam. Conidia hyaline, cylindrical, fusiformis, sometimes narrowed at the centre, (9.5-13.5) µm×(3-3.5) µm; mycelial appressoria dark brown, rounded, clavate, irregularly shaped, (5-8) µm×(3-5.5) µm. Interestingly, pathogenicity test showed that the C. camelliae could cause anthracnose lesions on the wounded leaves of ‘Longjing 43’ but not on the intact leaves; on the contrary, there were no visible disease symptoms on the ‘Zhongcha 108’ leaves. Moreover, both C. fructicola and C. siamense did not show pathogenicity on these two tea varieties. 【Conclusion】 Based on the phylogenetic analyses and the morphological observation of Colletotrichum strains isolated from tea plants from 15 provinces (municipality, autonomous region) of China, it is concluded that C. camelliae is the main causal agent of Ca. sinensis anthracnose in the major tea areas of China.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Effects of Low Molecular Weight Organic Acids on Soil Enzymes Activities and Bacterial Community Structure

YU Hui-yong, SONG Xiao-li, WANG Shu-sheng, CAO Li-jun, GUO Li, WANG Xiao-li, PENG Gong-yin

Scientia Agricultura Sinica. 2015, 48(24):  4936-4947.  doi:10.3864/j.issn.0578-1752.2015.24.008

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【Objective】Soil biological properties can reflect soil quality rapidly. The objective of this study is to clarify the effects of the low molecular weight organic acids on soil biological properties, and to provide a scientific basis for improving soil quality. 【Method】According to the amount of low molecular weight organic acids during tobacco early growth period and previous research results, a laboratory experiment was carried out to investigate the effects of six kinds of organic acids (benzoic acid, cinnamic acid, lauric acid, phthalic acid, myristic acid and palmitic acid) whose concentrations were 1, 2 and 3 g·kg^-1 (carbon/soil ratio) on four kinds of soil enzymes activities (soil catalase, alkaline phosphatase, urease and sucrase) and bacterial community structure. Tobacco field soil was cultivated in a dark environment at 25℃ for 30 d and the analysis methods for the bacterial community included the related evolutionary tree, principal co-ordinates analysis (PCoA), the classification of the relative abundance columnar diagram and the species abundance clustering heat maps, and so on. 【Result】Soil sucrase activity with three concentrations of cinnamic acid was lower than that of the control, cinnamic acid had the most inhibitory effect on soil sucrase activity. Soil urease activity with three concentrations of cinnamic acid, phthalic acid and benzoic acid were all lower than that of the control, their inhibitions were stronger. Cinnamic acid and benzoic acid had higher inhibitory effect on soil catalase activity, the value of benzoic with 3 g·kg^-1 concentration was the lowest. Cinnamic acid, phthalic acid and benzoic acid inhibited the soil alkaline phosphatase activity greatly whose three concentrations were lower than that of the control and benzoic acid with 3 g·kg^-1 was the lowest. The classification of the columnar diagram of different samples showed lauric acid with 2 g·kg^-1 concentration had bigger differences with the others, contrasted with the 6 others, the content of Cyanophyta, Chloroplast, Streptophyta and Streptophyta- unclassified were the highest at Phylum, Class, Order and Family levels separately, the analysis results were consistent with that of the related evolutionary tree and PCoA. The other 6 treatments had no obvious differences at above the Genus level and all the treatments had differences at the Genus level. 【Conclusion】The concentrations of low molecular weight organic acids had significant effects on soil enzyme activities, and soil bacteria may not be the main reason affecting soil enzyme activities.

Interactive Effects of Elevated CO₂ and Nitrogen on the Physiology and Yield of Winter Wheat in North Winter Wheat Region of China

JU Hui, JIANG Shuai, LI Jing-tao, HAN Xue, GAO Ji, QIN Xiao-chen, LIN Er-da

Scientia Agricultura Sinica. 2015, 48(24):  4948-4956.  doi:10.3864/j.issn.0578-1752.2015.24.009

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【Objective】This study aims to elucidate the interactive effects of elevated CO₂ concentration [CO₂] and different nitrogen applications on phenology, photosynthesis and yield of winter wheat, then to provide theoretical understanding for objective assessment on the potential yield of winter wheat under climate change conditions.【Method】The experiment was conducted from 2011-2014 using a Free Air CO₂ Enrichment (FACE) facility with pot experiment and winter wheat cultivar “Zhongmai 175”. Each pot was filled with 45 kg of sieved soil. The treatments were set at elevated [CO₂] of 550 mg·L^-1 and ambient [CO₂] of 390 mg·L^-1，nitrogen fertilizer applied at N1, 0.16g·kg^-1 soil and N0, 0 g·kg^-1 soil. The changes of phenology, physiology and yield of winter wheat were investigated. The elevated CO₂ feeding was in operation from regreen to maturity stage each year and injected time from 6:30-18:30 each day, and no CO₂ was released at night. The [CO₂] was controlled through computer program and could synchronously adjust with wind speed and direction time by a solenoid valve in a release pipe, then it achieved the expected [CO₂].【Result】The pot experiment results indicated that elevated [CO₂] accelerated the growth process of winter wheat compared with ambient [CO₂], jointing stage speeded up of 1 day, flowering period can be 1-2 days in advance, and the whole growth duration was shortened 3-5 days under the same fertilizer level. While as the high nitrogen could prolong the growth duration, the flowering period can be extended by 1-2 days, filling period by 4-5 days, synchronous alleviated the accelerative effects of elevated [CO₂] on growth process; High [CO₂] enhanced winter wheat yield about 16.0% via increased grain number per spike and grains weight significantly, and photosynthesis of flag leaves was enhanced by 13.7% compared with ambient [CO₂]. Under elevated [CO₂] conditions, higher application of nitrogen fertilizer could increase the yield of winter wheat about 50%，enhance the photosynthesis increase rate about 2.5% and transpiration about 13.5% compared with low nitrogen fertilizer supply. That means the nitrogen fertilization can not only promote the high [CO₂] positive effect, but still plays a more important role to maintain a stable and higher yield of winter wheat even with [CO₂] enrichment conditions; Moreover, elevated [CO₂] can increase the grain number per spike by 3.69% compared with ambient [CO₂], higher nitrogen increased by 3.43% compared with low nitrogen, the dual role of high nitrogen and high [CO₂] increase grain number highest of 38.37 grains/ear, low nitrogen and ambient [CO₂] treatment had the lowest level of spike grain numbers, which indicated high [CO₂] has the slightly higher positive effects than that of a purely nitrogen treatment on grain number. Dual effects of high [CO₂] and nitrogen had the most positive effects on grain number, but the effects had no obvious difference if these measures are used separately. High [CO₂] increased grain weight by 5.3%, and if it combined with high nitrogen that the grain weight could increase 7.3%. All these showed that nitrogen promoted the positive effects of high [CO₂] on grain weight.【Conclusion】Therefore, under future climate change conditions, higher [CO₂] has positive effects to enhance yield of winter wheat and has obvious positive interactions with nitrogen, high nitrogen can extend the growth period which mitigate the shorten growth duration by higher [CO₂], improve photosynthesis and promote CO₂ fertilization effects. Higher [CO₂] could increase yield via high grain number per spike and grains weight which could be considered for wheat breeding.

HORTICULTURE

Effects of Boron Deficiency on Cellular Structures of Maturation Zone from Root Tips and Functional Leaves from Middle and Upper Plant in Trifoliate Orange Rootstock

LIU Lei-chao, JIANG Cun-cang, DONG Xiao-chang, WU Xiu-wen, LIU Gui-dong, LU Xiao-pei

Scientia Agricultura Sinica. 2015, 48(24):  4957-4964.  doi:10.3864/j.issn.0578-1752.2015.24.010

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【Objective】The aim of this study was to determine the effects of boron deficiency on the cellular structures of the maturation zone in root tips and the functional leaves in citrus. 【Method】A hydroponic experiment was conducted with (10 μmol·L-1 H3BO3, +B) and without boron (-B) treatment. Fine root mature zone and young leaf samples were observed by paraffin sections and transmission electron microscope (TEM). 【Result】Boron deficiency reduced the amount of parenchyma cell in root and, arranged loosely, led to cell deformation and enlarged the intercellular space. Also, boron deficiency caused the disintegration of the cytoplasm and organelles, and increased the cell wall. Whereas the parenchyma cell morphology was normal, evenly sized, and arranged densely in +B treatment, the parafin section showed the root vascular bundle was dyed deeper and the structure was clearly organized in a circular pattern around the root pulp. Low B stress seriously inhibited the development of vascular bundle and restricted its differentiation, making the cell small and randomly arranged. In the plant, Boron leakage made the leaves thick and uneven and sponge tissue became deformed, leading to bigger cell gaps and an increase in the number and volume of sponge tissue. This improved the proportion of sponge tissue in mesophyll. However, in B+ treatment, leaf thickness was more even and the epidermis cell was more densely arranged. The palisade mesophyll was closely packed and sponge tissue underneath was loosely and organically arranged. Under B deficiency, there was also an accumulation of starch grains in the cell and leaf soluble sugar and starch content increased by 35.3% and 66.7%, respectively. But there was no obvious accumulation of starch in the leaves with +B treatment. 【Conclusion】 The internal structure of the leaf cell was destroyed by boron deficiency. Cell wall thickness and starch accumulation intensified while sponge tissue showed obvious proliferation. The leaf’s abnormal growth further inhibited the development of leaf and root vascular bundles.

Comparative Proteomic Analysis in Peels of Chuntianju (Citrus reticuiata Blanco) and Its Mutant

ZENG Ji-wu, DENG Gui-ming, GAO Chang-yu, JIANG Bo, ZHONG Yun, ZHONG Guang-yan, YI Gan-jun

Scientia Agricultura Sinica. 2015, 48(24):  4965-4978.  doi:10.3864/j.issn.0578-1752.2015.24.011

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【Objective】 Bud mutation of citrus is one of the most important breeding technique for new cultivars. However, little is known about the mechanisms of citrus bud mutation, particularly at the molecular level. Mingliutianju (C. reticuiata Blanco cv. Mingliutianju, MP) is a new cultivar selected from Chuntianju (C. reticuiata Blancocv. Chuntianju, CP) bud mutation. To identify the differentially expressed proteins in peels of Chuntianju (C. reticuiata Blanco cv. Chuntianju, CP) and its bud mutant Mingliutianju (C. reticuiata Blanco cv. Mingliutianju, MP) and to clarify the possible mechanism of this phenotype bud mutation. 【Method】Two-dimensional gel electrophoresis (2-DE) combined with the mass spectrometry (MS) technology were applied to separate the differentially expressed proteins (DEPs) between the MP and CP using peels at 12 weeks and 23 weeks after flowering. Meanwhile, the identified differentially expressed proteins (DEPs) with fold-change of more than 2 were analyzed using MALDI-TOF-MS and Bioinformatics. 【Result】 Clear 2-DE gel maps of peels of both CP and MP were successfully obtained. In total, more than 1200 protein spots and 500 protein spots were detected by silver stained (SS) 2-DE gels and by Coomassie brilliant blue stained (CS) 2-DE gels respectively. Forty DEP spots (20 identified from SS 2-DE gels and 20 from CS 2-DE gels) were further analyzed through MALDI-TOF-MS. Totally, 33 annotated DEPs, including 17 up-regulated and 16 down-regulated DEPs, were identified in MP. Gene ontology analysis revealed that these 33 DEPs could be divided into 11 categories including carbohydrate/energy metabolism, stress/defense response, nucleotide metabolism, amino acid metabolism, transcription, nitrogen metabolism, lipid metabolism, unclassified, protein modification and degradation and unknown pathways. Moreover, by using the web-based platform KOBAS (KEGG Ontology-Based Annotation System), 18 of the 33 DEPs were successfully mapped to 31 KEGG pathways, among which the flavonol biosynthetic process pathway was ranked the first. 【Conclusion】 Intensive studies were conducted to identify the mechanism that lead to the morphological differences between CP and MP from both the gene expression and the protein expression levels. Results generated in this study suggested that the flavonol biosynthetic process play an essential role in CP’s spontaneous bud mutation process. Notably, the differential expression of 2 UDP-glucosyltransferase genes and 1 caffeoyl-CoA O-methyltransferase protein may contribute a lot to the morphological differences.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Effects of Weaning Stress on Growth Performance, Organ Development and Serological Parameters in Lambs

CHAI Jian-min, WANG Hai-chao, DIAO Qi-yu, QI Min-li, GUO Feng, WANG Hai-chao*, ZHANG Nai-feng

Scientia Agricultura Sinica. 2015, 48(24):  4979-4988.  doi:10.3864/j.issn.0578-1752.2015.24.012

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【Objective】To investigate the effects of weaning time on growth performance, nutrient digestibility, organ development and serum parameters in Hu sheep lambs for confirming best weaning age, seventy-two lambs of Hu sheep were divided into four groups with similar birthday and birth weight with 16 lambs in each experimental group and 24 lambs in the control group（ER group. 【Method】The lambs in experimental groups were weaned at 10, 20 or 30 days of age (EW10, EW20, EW30 groups) by feeding milk replacer, and the lambs in control group were ewe-reared. The digestion trial was carried out within 10 days after the weaning day, and the growth performance, organs development, serum parameters were determined on 10th day after weaning day. At the same time, lambs in ER group were disposed uniformly. 【Result】 The statistical findings were as follows: (1) body weight and average daily gain in EW10 and EW30 but not EW20 were significantly lower than that in ER (P＜0.05; (2) the starter dry matter intake in EW10 and EW20 but not EW30 were significantly higher than that in ER (P＜0.05; (3) during the 10 days after weaning, nutrient digestibility of dry matter (DM) and organic matter (OM) in EW10 group showed no significant difference with ER group (P＞0.05), whereas nutrient digestibility of nitrogen (N), gross energy (GE), ether extract (EE), calcium (Ca) and phosphorus (P) in EW10 group was lower than that in ER group (P＜0.05); (4) in addition, the nutrient digestibility included DM, OM, N, GE, EE, Ca and P in EW20 and EW30 groups were significantly lower than ER (P＜0.05); (5) the ratio of organs to body weight between groups showed no significant differences except in the ratio of rumen weight to body weight between EW10 and ER (P＜0.05); (6) serum parameters showed no significant difference at day of weaning (P＞0.05). However, on the 10th day post-weaning, EW10 and EW30 had a significantly lower TP and ALB and a higher CORT than ER (P＜0.05), and (7) the TNF-α in EW10 were higher than ER. 【Conclusion】 Weaning at 20 days of age showed minimum weaning stress according to the performance of weaned lambs.

Study on Expression of Glutathione Peroxidase type-5 (GPX5)   and Protein Localization in Epididymis of Adult Sheep

LI Rui-lan, ZHANG Tong, FAN Xiao-mei, OU YANG Xiao-qing, ZHANG Chun-qiang, CAO Jun-wei, ZHANG Jia-xin

Scientia Agricultura Sinica. 2015, 48(24):  4989-4995.  doi:10.3864/j.issn.0578-1752.2015.24.013

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【Objective】 The objective of this study was to investigate the expression pattern of GPX5（Glutathione peroxidase type-5）and GPX5 protein localization in adult sheep epididymis to provide a theoretical basis for studying antioxidative mechanisms of sperm in sheep epididymis. 【Method】 Epididymis, testis and deferent duct of adult Mongolia sheep of similar age were collected. The epididymis of each ram were selected as epididymal caput, epididymal corpus and epididymal cauda, respectively. Samples were stored at -80 ℃. The expression of GPX5 in epididymis, testis and deferent duct were detected by real-time fluorescent quantitative PCR and Western blot. The fresh tissue samples of different segments were cut to suitable size and immersed in 4% paraformaldehyde solution for 24 hours; then paraffin embedded tissue sections were made and incubated with GPX5 specific antibody. The positive signals of GPX5 protein were detected by DAB kit. 【Result】The result of real-time fluorescent quantitative PCR showed that the expression level of GPX5 of caput was significantly higher than that of corpus and cauda (P＜0.01), but almost no expression were found in testis or deferent duct. We showed that GPX5 protein was highly expressed in caput, and a small amount of GPX5 protein was present in corpus and cauda; however no GPX5 protein was found in testis nor deferent duct. Collectively, these studies suggested that GPX5 expressed mainly in the epididymal caput of adult sheep. As assessed by immunohistochemical staining, the GPX5 protein was localized mainly in the epithelium and stereocilia of caput, while in corpus and cauda the GPX5 protein localized mainly in stereocilia. The GPX5 protein was detected in sperm located in caput, corpus, cauda as well as epididymal lumen. The GPX5 protein secreted from epididymis epithelium into epididymal lumen and combined gradually with spermatozoa during epididymal transit. 【Conclusion】GPX5 was synthesized and secreted mostly in the epididymal caput epithelium of adult sheep and combined with spermatozoa in epididymal lumen, thereby providing protection for their normal physiological functions.

Establishment of a GeXP Analyser-Based Multiplex PCR Assay for Detection of Eight Reproductive and Respiratory Swine Pathogens

ZHANG Min-xiu, XIE Zhi-xun, DENG Xian-wen, XIE Zhi-qin, XIE Li-ji, HUANG Li, HUANG Jiao-ling

Scientia Agricultura Sinica. 2015, 48(24):  4996-5006.  doi:10.3864/j.issn.0578-1752.2015.24.014

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【Objective】A GeXP-multiplex PCR assay was developed to simultaneously detect eight swine reproductive and respiratory viruses, including reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus 2 (PCV2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). 【Method】Nine pairs of specific primers were designed based on the conserved sequences of eight pathogens available in the GenBank database. Each gene-specific primer was fused at the 5’ end of a universal sequence to generate 9 pairs of specific chimeric primers. The GeXP-single PCR assay was performed using a single cDNA/DNA template to determine the feasibility of the primers. The GeXP-mutiplex PCR assay was performed using a single cDNA/DNA template and 9 pairs of the specific chimeric primers to determine the specificity and accuracy of the GeXP-mutiplex PCR assay. Serial dilution from 103 copies/μL to 10 copies/μL of in vitro transcripted RNA of target genes (SIV-H1, SIV-H3, PRRSV, CSFV and JEV) and plasmid of PCV-2, PPV and PRV were used to examine the single template sensitivity of GeXP multiplex PCR assay; Serial dilution from 104 copies/μL to 10 copies/μl of all viral targets were used to examine the sensitivity of eight pathogens of GeXP multiplex PCR assay. The GeXP assay was evaluated using 23 clinical specimens and compared with the single PCR assay. 【Result】The corresponding specific fragments of genes were amplified by the GeXP -single PCR assay and the GeXP-mutiplex PCR assay. The results confirmed the feasibility of the primers and the specificity and accuracy of the GeXP assay. The GeXP assay achieved a sensitivity of 10 copies/µL for a single pathogen. The sensitivity of the GeXP assay to detect the eight pathogens simultaneously was 103 copies/µL. The GeXP assay successfully detected twenty-three clinical samples and the results of the GeXP assay were consistent with the results by the single PCR assay.【Conclusion】The GeXP assay was established successfully for simultaneously detecting eight swine reproductive and respiratory pathogens; These results showed that the GeXP assay is a specific, sensitive and high-throughput test to detect swine reproductive and respiratory pathogens. This assay provides a new test for the simultaneous differentiation of eight swine reproductive and respiratory diseases.