应用Illumina MiSeq高通量测序技术分析河西走廊地区盐碱土壤微生物多样性

【目的】以甘肃省河西走廊地区的9个盐碱土壤样品(原生盐碱土、次生盐碱土、农田土)为材料,研究该地区盐碱土壤中微生物群落的多样性。【方法】提取土壤微生物总DNA,应用Illumina Mi Seq高通量测序技术进行分析。【结果】从分布在河西走廊3个流域的9个盐碱土样品中共获得325 089条微生物的16S r RNA基因序列。冗余分析和热图分析表明,原生盐碱土与次生盐碱土、原生盐碱土与农田土微生物群落构成差异较大,次生盐碱土与农田土微生物群落差异较小。土壤p H对微生物群落组成的影响最显著。多样性指数和稀释性曲线分析得出,在9个土壤样品中,S6号Shannon指数最大,S1号Shannon指数最小,S1号Simpson指数最大,S6号Simpson指数最小,说明原生盐碱土的微生物群落多样性最低,次生盐碱土的微生物群落多样性最高。盐碱土壤中主要的微生物群落包括9个门,其中变形菌门占主导地位,其余依次是放线菌门、拟杆菌门、酸杆菌门、浮霉菌门、绿弯菌门、芽单胞菌门、厚壁菌门和疣微菌门。原生盐碱土和农田土中占优势的微生物群落是变形菌门,次生盐碱土中占优势的微生物群落是放线菌门。【结论】河西走廊地区盐碱土壤中微生物多样性非常丰富,存在大量的微生物类群,尤其是在次生盐碱土壤中。     

大兴安岭典型永久冻土土壤细菌群落组成和多样性

【背景】土壤微生物是土壤生物中的重要成分,参与了土壤生态系统中关键的生物化学循环过程。但是关于寒温带多年冻土土壤微生物的研究还比较薄弱。【目的】探究大兴安岭多年冻土土壤中微生物的多样性和种群结构。【方法】利用MiSeq高通量测序技术对黑龙江大兴安岭地区呼中保护区落叶松冻土和樟子松林冻土土壤样品进行测序。【结果】在落叶松冻土和樟子松林冻土土壤中,相对丰度最高的优势菌群的组成基本一致,在门水平有疣微菌门(Verrucomicrobia)、变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、浮霉菌门(Planctomycetes)、绿菌门(Chlorobi)、Parcubacteria、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、芽单胞菌门(Gemmatimonadetes)10个细菌门类,其中,疣微菌门(Verrucomicrobia)在樟子松林土壤中的相对丰度较多,变形菌门(Proteobacteria)在落叶松林土壤中的相对丰度较多。通过α多样性分析可知,落叶松冻土土壤微生物的群落多样性高于樟子松林冻土,而且两者的细菌群落组成与结构差异性较大。【结论】为深入认识大兴安岭多年冻土区的土壤微生物群落结构组成以及影响因素提供数据支撑。     

塔里木河中下游流域棉田及胡杨林土壤细菌群落结构及多样性研究

【背景】新疆绿洲生态系统的大规模农垦开发对当地生态环境产生了一系列影响,评估农业活动对新疆地区原始生态土壤细菌群落的影响对合理利用土地、开发农业资源具有指导意义。【目的】通过研究塔里木河中下游流域受农垦开发影响的棉田及未经历土地开垦的胡杨林土壤细菌群落结构及多样性的变化,探究农垦开发活动对土壤微生态的影响。【方法】利用国标法测定塔里木河中下游流域绿洲棉田及原始胡杨林全氮、总盐、有机质、碱解氮、有效磷、速效钾、pH等土壤理化性质;提取土壤总DNA,通过PCR扩增建立文库,用新一代Illumina HiSeq 2500测序技术对土样DNA进行16SrRNA基因(V4区)高通量测序;利用生物信息学分析细菌群落的α多样性(包括Chao1、Observed species、Phylogenetic diversity (PD)、Shannon、Simpson和Good’s coverage指数)、β多样性在两地的变化,基于线性判别分析流程[Lineardiscriminantanalysis(LDA)effectsizepipeline,LEfSe]分析在两地具有显著差异的细菌分类单元;结合土壤理化参数、群落功能预测分析可能影响两地细菌群落结构变化的主要因子。【结果】土壤基本理化性质分析:绿洲棉田与原始胡杨林相比,全氮、碱解氮、总盐、pH含量显著增加,土壤盐含量与全氮、碱解氮和pH呈显著正相关(P0.05);细菌多样性分析:α多样性指数Chao1、Observedspecies和PD在棉田样品中均显著低于胡杨林样品,Shannon指数差异虽不显著,但是也表现出同样的变化趋势;细菌群落组成分析:两组样品中的优势细菌依次为α-变形杆菌纲、放线菌门、芽单胞菌门、拟杆菌门、酸杆菌门、γ-变形菌纲、β-变形菌纲、δ-变形菌纲、绿弯菌门、厚壁菌门、浮霉菌门、泉古菌门和硝化螺旋菌门(丰度1%);差异分析的结果显示,新疆绿洲棉田土壤中显著富集的微生物主要包括酸杆菌门、放线菌门、绿弯菌门、厚壁菌门、黄杆菌纲、α-变形菌纲、γ-变形菌纲中的一些物种,以及泉古菌门中的Candidatus Nitrososphaera;新疆原始胡杨林土壤中显著富集的微生物主要包括绿菌门、δ-变形菌纲以及变形菌门中的其他物种,还包括泉古菌门中的Nitrosopumilus。主坐标分析(PCoA)结果显示,棉田生态系统的土壤细菌群落结构能够与胡杨林生态系统的群落结构明显分开,且前者的细菌群落结构一致性更高。功能预测结果显示,绿洲棉田的硝化作用显著高于原始胡杨林。冗余分析(RDA)结果表明全氮含量对细菌群落结构有显著影响。【结论】在长期耕种施肥等人为因素干扰下,绿洲棉田土壤细菌多样性和群落结构发生明显改变,细菌α多样性明显低于原始胡杨林,细菌β多样性差异减弱,同时一些与植物生长密切相关的细菌类群,如放线菌、硝化细菌等显著富集。该研究可为新疆绿洲棉田生态系统的开发利用现状提供数据参考。     

高通量测序分析大兴安岭典型森林土壤细菌和真菌群落特征

【背景】大兴安岭地区是我国对气候变化响应最敏感的区域,而土壤微生物在维持寒区森林生态系统结构和功能中发挥重要作用。【目的】探究大兴安岭不同森林类型土壤微生物群落结构及与环境因子的关系。【方法】采用Illumina MiSeq高通量测序技术,分析3种典型森林(白桦林、落叶松林和樟子松林)土壤细菌和真菌群落组成和多样性。【结果】3种林型中共获得2 786个细菌操作分类单元(Operational Taxonomic Unit,OTU),隶属于38门531属,其中优势菌门为变形菌门(Proteobacteria, 31.45%-40.32%)、酸杆菌门(Acidobacteria, 14.24%-40.16%)和放线菌门(Actinobacteria,7.13%-22.15%);1803个真菌OTU隶属于8门263属,其中优势菌门为担子菌门(Basidiomycota,40.43%-62.75%)和子囊菌门(Ascomycota,35.81%-53.68%)。主坐标分析表明,3种林型中细菌和真菌群落组间差异远大于组内差异。Mantel检验结果显示:细菌群落结构与pH、总氮(Total Nitrogen,TN)、总磷(Total Phosphorus,TP)和含水量(Soil Water Content,SWC)具有显著相关性(P0.05),其中pH的相关性系数最大;真菌群落结构与SWC、TN和TP具有显著相关性(P0.05),其中TN的相关性系数最大。冗余分析结果发现,TP与变形菌门、担子菌门相对丰度呈显著正相关,TN和SWC与酸杆菌门、子囊菌门呈显著正相关,pH与放线菌门呈显著正相关(P0.05)。【结论】不同林型间土壤微生物群落结构存在显著差异,明晰其分布规律及主要环境驱动因子,是把握寒区森林生态系统过程的关键。     

湿地法处理生活污水对仙桃湿地地表水微生物群落结构的影响

【背景】随着中小城市经济的高速发展和人们生活水平的提高,生活污水排放量也日益增大,致使水源水质污染呈恶化趋势。【目的】了解湿地法处理生活污水对湿地地表水微生物群落多样性变化的影响。【方法】对人工湿地(仙桃)参照点(-2 000)、200、400、600、3 000、5 000 m水平方向6点地表水进行理化检测,再通过高通量测序对微生物群落结构进行解析,并探讨微生物群落结构变化与生活污水的污染效应。【结果】当湿地法处理的生活污水到达5 000 m时,化学需氧量、氨态氮、总氮和总磷均达《城镇污水处理厂污染物排放标准》一级标准,且理化指标之间均呈极显著正相关。样品在门水平上的优势物种分别为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、蓝藻门(Cyanobacteria)、疣微菌门(Verrucomicrobia)、厚壁菌门(Firmicutes)、酸杆菌门(Acidobacteria)和绿弯菌门(Chloroflexi)。变形菌门(Proteobacteria)可用来作为排放生活污水污染物的指示性微生物,放线菌门(Actinobacteria)、蓝藻门(Cyanobacteria)、疣微菌门(Verrucomicrobia)和绿弯菌门(Chloroflexi)可用来作为排放生活污水净化的指示性微生物。【结论】首次比较分析了湿地法处理仙桃生活污水地表水环境中微生物群落多样性变化的情况,可使人们从微生物层面更加明确地了解人工湿地运作机理及地表水环境的污染情况。     

16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

【目的】通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据。【方法】通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成。【结果】转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异。【结论】通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限。     

韭菜迟眼蕈蚊体内及韭菜根际微生物的多样性分析

【目的】为了了解韭菜迟眼蕈蚊Bradysia odoriphaga生存环境中微生物的多样性。【方法】本研究对韭蛆、韭菜鳞茎、根部以及土壤中的微生物进行分离纯化,对部分代表性细菌进行了生理生化特征的分析,并对分离得到的真菌及细菌分别采用ITS和16S r DNA基因分析的方法进行鉴定。【结果】总共分离鉴定出25种微生物,在韭菜鳞茎和土壤中分别分离出半知菌亚门(Deuteromycotina)的球孢白僵菌Beauveria bassiana和球孢虫草Cordyceps bassiana两种真菌,在韭蛆体内、韭菜鳞茎、韭菜根部以及土壤中分别分离出10、15、9、6种细菌,以变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)细菌为主,其余的细菌分别属于放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)。【结论】本研究可以为寻找韭菜、韭蛆和微生物三者之间的协同进化关系提供理论基础,并期望为韭蛆的共生防治提供指导。     

长期施肥土壤微生物群落的剖面变化及其与土壤性质的关系

【目的】认识不同施肥模式对土壤微生物群落的长期影响及其与土壤理化属性的联系。【方法】利用新一代高通量测序技术,研究绿洲农田20年单施化肥(N 300 kg/hm2、P2O5150 kg/hm2与K2O 60 kg/hm2)与化肥配施秸秆(同量的N与P肥配施5.4 t秸秆)对土壤剖面(0-300 cm)微生物群落结构的影响。【结果】放线菌与α-变形菌为土壤表层(0-20 cm)的优势类群。随土壤剖面深度的增加,放线菌相对丰度减少,而变形菌,特别是γ-变形菌与β-变形菌相对丰度增加,逐渐成为深层(20-300 cm)土壤中的优势类群。长期施肥对整个土壤剖面的微生物群落结构均有显著影响,并且明显提高了0-40 cm土层中氨氧化古菌的相对丰度。此外,农田管理模式如灌溉可能是氨氧化细菌在土壤垂直剖面的重要驱动因素。统计分析表明土壤全氮含量对表层土壤中微生物群落结构的影响最大,而有机碳含量则是影响深层土壤微生物群落的最重要因子。【结论】长期施肥改变了土壤剖面碳源与氮源的可利用量,导致了施肥处理间土壤微生物群落结构的差异,特别在剖面深层更为明显。     

长期施肥土壤微生物群落的剖面变化及其与土壤性质的关系

摘要:【目的】认识不同施肥模式对土壤微生物群落的长期影响及其与土壤理化属性的联系。【方法】利用新一代高通量测序技术,研究绿洲农田20年单施化肥(N 300 kg/hm2、P2O5 150 kg/hm2与K2O 60 kg/hm2)与化肥配施秸秆(同量的N与P肥配施5.4 t秸秆)对土壤剖面(0－300 cm)微生物群落结构的影响。【结果】放线菌与α-变形菌为土壤表层(0－20 cm)的优势类群。随土壤剖面深度的增加,放线菌相对丰度减少,而变形菌,特别是γ-变形菌与β-变形菌相对丰度增加,逐渐成为深层(20－300 cm)土壤中的优势类群。长期施肥对整个土壤剖面的微生物群落结构均有显著影响,并且明显提高了0－40 cm土层中氨氧化古菌的相对丰度。此外,农田管理模式如灌溉可能是氨氧化细菌在土壤垂直剖面的重要驱动因素。统计分析表明土壤全氮含量对表层土壤中微生物群落结构的影响最大,而有机碳含量则是影响深层土壤微生物群落的最重要因子。【结论】长期施肥改变了土壤剖面碳源与氮源的可利用量,导致了施肥处理间土壤微生物群落结构的差异,特别在剖面深层更为明显。     

枯草芽胞杆菌菌肥对有机冬瓜根区土壤微生态的影响

【背景】微生物肥料已广泛应用于我国有机作物的种植,其对有机种植土壤微生态的影响尚需科学评测。【目的】高通量测序技术可用于精确分析土壤微生物群落,从细菌、真菌群落结构和多样性的角度阐释枯草芽胞杆菌菌肥对有机农田根区土壤微生物群落的影响。【方法】在有机农田轮作种植条件下,施用枯草芽胞杆菌菌肥后提取冬瓜根区土壤基因组DNA,通过PCR扩增建立文库,利用IlluminaMiSeq高通量测序技术,并结合相关生物信息学方法分析土壤细菌16SrRNA基因V3-V4区和真菌ITS1区的多样性指数及群落结构;测定根区土壤化学性质及酶活性,分析有机冬瓜果实品质,并作相关分析。【结果】从6个有机冬瓜根区土壤样本中获得14199个细菌操作分类单元(OTU)和3378个真菌OTU,细菌和真菌文库测序覆盖率分别在98%、99%以上。枯草芽胞杆菌菌肥会在一定程度上提高土壤细菌种群多样性而降低真菌种群多样性,丰富了细菌群落结构,但显著降低了真菌群落丰富度(P0.05);并减少了根区土壤特有细菌和真菌物种。变形菌门、厚壁菌门和放线菌门是优势细菌,子囊菌门是优势真菌;枯草芽胞杆菌菌肥会提高绿弯菌门和子囊菌门的相对丰度,比例分别为46.23%、10.01%;降低变形菌门和担子菌门的相对丰度,比例分别为11.14%、74.72%。枯草芽胞杆菌菌肥显著降低了土壤pH,显著提高了有机冬瓜果实总氨基酸、可溶性固形物等营养成分含量(P0.05)。【结论】施用枯草芽胞杆菌菌肥改变有机冬瓜根区土壤细菌和真菌的丰富度和多样性,降低了土壤pH,提高了有机冬瓜果实品质。     

影响水稻光合日变化的酶和相关因素的分析

水稻叶片的最大光合速率出现在上午１０：００时,Ｒｕｂｉｓｃｏ初始活力也在此时达到最大,然后逐渐降低,下午１４：００时略上升后又下降．Ｒｕｂｉｓｃｏ初始活力与光合速率之间极显著相关,相关系数为 ０．９４７４．运用相关性分析、回归分析、通径分析,对晴（有时有云）天气水稻叶片的光合速度、光合关键酶及有关因素的日变化进行综合评估,结果发现中午光合下降主要来自气孔限制,同时Ｒｕｂｉｓｃｏ活性也下降;Ｒｕｂｉｓｃｏ活性是影响光合日变化的又一重要生化因子．体内Ｒｕｂｉｓｃｏ活性受Ｒｕｂｉｓｃｏ活化酶的调节．     

甲醇对酵母过氧化氢酶活性的影响机理研究

将酵母过氧化氢酶加入一定比例的甲醇,测定其活性变化。结果表明:在含2%甲醇时酶活比对照提高4026%。将粗酶液用70%饱和度的硫酸铵盐析后离心所得的上清液再加入硫酸铵至80%饱和度,离心的沉淀溶解在缓冲液中,上SephadexG75柱,分离出的有酶活性的蛋白峰经电泳得一条蛋白带,说明过氧化氢酶已经被提纯到电泳纯。光谱分析发现,甲醇处理后过氧化氢酶纯酶的吸收光谱和荧光发射光谱与未经处理的比较基本不变,而差示光谱出现明显的正峰和负峰。由动力学分析可知,在甲醇中,过氧化氢酶的Vmax和Km值均有不同程度提高 。     

凡纳滨对虾各月龄性状的主成分与判别分析

为了研究凡纳滨对虾各性状增长规律和判定最佳生长季节凡纳滨对虾的体格与月龄的关系,选择1～6月龄凡纳滨对虾各1000只,选择全长、体长、第一腹节背高、第三腹节背高、第一腹节背宽、头胸甲长和体重共7个性状,进行主成分与判别分析.结果表明:各月龄凡纳滨对虾性状之间均呈现显著的正相关(P<0.01), 其中以全长与体长的相关性最为明显,1月龄凡纳滨对虾体重与形态性状的相关系数较小.各月龄凡纳滨对虾的主成分有所不同,1～2月龄凡纳滨对虾的第一主成分为长度因子,第二主成分为宽度因子,第三主成分为高度因子;3月龄凡纳滨对虾的第一主成分与1～2月龄凡纳滨对虾一致,但第二主成分为高度因子,第三主成分为体重因子;4～6月龄凡纳滨对虾的第一主成分为体重因子,第二主成分为高度因子,第三主成分为宽度因子.1～3月龄凡纳滨对虾形态性状的增长优先于体重, 4～6月龄凡纳滨对虾体重优先于形态性状的增长.错过最佳生长季节的凡纳滨对虾的与体格大小相符的月龄可通过建立的判别式来判断,总的判别准确率为98.98%,其中2～4月龄凡纳滨对虾的判别准确率为100%.     

Discrimination of higher plant calluses based on embryogenic capacity and taxonomic classification by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum, and has been widely applied to the discrimination of closely related microbial strains. Minimally prepared samples of embryogenic and non-embryogenic calluses derived from various higher plants (sweet potato, morning glory, Korean ginseng, Siberian ginseng, and balloon flower) were subjected to PyMS for spectral fingerprinting. A dendrogram based on the unweighted pair group method, with arithmetic mean of pyrolysis mass spectra, divided the calluses into Siberian ginseng embryogenic callus and the others, which were subsequently divided into embryogenic and non-embryogenic callus groups, regardless of plant species from which the calluses were derived. In the non-embryogenic callus group, the dendrogram was in agreement with the known taxonomy of the plants. These results indicate that PyMS analysis could be applied for discriminating plant calluses based on embryogenic capacity and taxonomic classification.     

Normal Approximations of Exact Tests in Configural Frequency Analysis

The most common tests for types and antitypes in configural frequency analysis are normal approximations of exact tests. In the paper such statistics under the complete independence model and under the fixed margins model are discussed. It turns out that these test statistics are not acceptable when the number of simultaneously performed tests is large or when the expected frequencies are small. In these cases, the use of exact tests is advocated and some existing computer programs for such tests are indicated. A normal approximation based on the strong version of the De Moivre-Laplace limit theorem is also discussed. Empirical examples are given from longitudinal data describing psychological development of boys.     

Some remarks about the analysis of active control studies

In an active-controlled trial, the experimental treatment can be declared to be non-inferior to the control if the confidence interval for the difference excludes a fixed pre-specified margin. Recently, some articles have discussed an alternative method where the data from the current study and placebo-controlled studies for the active control are combined together into a single test statistic to test whether a fixed fraction of the effect of the active control is preserved. It has been shown that, conditional on nuisance parameters from the active-controlled study, a fixed margin can be defined that will be operationally equivalent to this latter method. In this article, we will discuss statistical properties associated with these approaches. Specifically, the interim monitoring boundaries and level of evidence will be considered.     

Phenotypic evaluation of genetic variability and selection of yield contributing traits in chickpea recombinant inbred line population under high temperature stress

Heat is a major abiotic stress that drastically reduces chickpea yield. This study aimed to identify heat-responsive traits to sustain crop productivity by screening a recombinant inbred line (RILs) population at two locations in India (Ludhiana and Faridkot). The RIL population was derived from an inter-specific cross between heat-tolerant genotype GPF 2 (C. arietinum L.) and heat sensitive accession ILWC 292 (C. reticulatum). The pooled analysis of variance showed highly significant differences for all the traits in RILs and most of the traits were significantly affected by heat stress at both locations. High values of genotypic coefficient of variation (19.52–38.53%), phenotypic coefficient of variation (20.29–39.85%), heritability (92.50–93.90%), and genetic advance as a percentage of mean (38.68–76.74%) have been observed for plant height, number of pods per plant, biomass, yield, and hundred seed weight across the heat stress environments. Association studies and principal component analysis showed a significant positive correlation of plant height, number of pods per plant, biomass, hundred seed weight, harvest index, relative leaf water content, and pollen viability with yield under both timely-sown and late-sown conditions. Path analysis revealed that biomass followed by harvest index was the major contributor to yield among the environments. Both step-wise and multiple regression analyses concluded that number of pods per plant, biomass and harvest index consistently showed high level of contribution to the total variation in yield under both timely-sown and late-sown conditions. Thus, the holistic approach of these analyses illustrated that the promising traits provide a framework for developing heat-tolerant cultivars in chickpea.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00977-5.     

Comparison of the Halving of Tablets Prepared with Eccentric and Rotary Tablet Presses

The aim of this study was to compare the densification of powder mixtures on eccentric and rotary tablet presses and to establish relationships with the halving properties of the resulting scored tablets. This is an important problem because the recent guidelines of EU require verification of the equal masses of tablet halves. The models of Walker, Heckel, and Kawakita were used to describe the powder densification on the two machines. The calculated parameters revealed that the shorter compression cycle of rotary machines results in poorer densification and lower tablet hardness at a given compression force. This is manifested in poorer halving properties, which are influenced mainly by the hardness. Better densification improves the halving even at lower tablet hardness. This demonstrates that these parameters can be good predictors of tablet halving properties.     

Fish ecoregions of Kansas: stream fish assemblage patterns and associated environmental correlates

Synopsis Principal components analysis was performed on fish presence/absence data for 39 common fish species from 410 stream sites in Kansas. The analysis confirmed ten ecologically meaningful fish assemblages, based on species associations. Factor scores based on these assemblages were then clustered into six geographic areas or fish ecoregions. Canonical discriminant analysis identified environmental variables that distinguished the derived fish ecoregions. Mean annual runoff, mean annual growing season, and discharge appear most important. Mean width, mean depth, chloride concentration, water temperature, substrate type, gradient, and percent of pool habitat were less important. Correspondence exists between these fish ecoregions and the patterns of physiographic regions, river basins, geology, soil, and potential natural vegetation in Kansas. The multivariate statistical approach used to classify fish ecoregions should have considerable potential value for fish assessment and management purposes in areas other than the state of Kansas.