黄瓜SRAP反应体系的正交设计优化

采用正交试验设计方法,对影响黄瓜SRAP反应体系的5种因素(dNTP、模板DNA、引物、Taq聚合酶及变性剂)4个水平进行优化筛选,确立了适合黄瓜SRAP分析的优化反应体系,即在10μL PCR反应体系中含有1μL 10×PCR buffer,150μmol/L dNTP,30 ng模板DNA,0.3μmol/L引物、1.5 U Taq聚合酶,PCR产物变性时用10μL变性剂。     

石斛属植物ISSR扩增体系的建立与优化

旨在建立并优化石斛属植物的ISSR-PCR反应体系。通过对ISSR-PCR反应体系中主要影响因子模板DNA、dNTP、10×PCR Buffer、引物以及Taq DNA聚合酶分别进行单因素优化,最终建立一套适用于石斛属植物的扩增多态性高、稳定性强、带型清晰的ISSR最佳反应体系。20μL反应体系的适宜浓度及用量分别为：模板DNA 10 ng/μL 3μL,dNTP 2.5 mmol/L 1.5μL,10×PCR Buffer 3.0μL,引物4μmol/L 3.5μL,TaqDNA聚合酶5 U 0.2μL。这一优化体系适用于石斛ISSR分析,为今后遗传多样性与亲缘关系分析、连锁图谱构建、QTL定位、基因定位与克隆等方面的研究提供了技术支撑。     

黄瓜叶片直接PCR扩增方法的建立

为了快速对黄瓜样品进行基于PCR的分子鉴定,本研究以我国华北型黄瓜品种"长春密刺"的幼嫩叶片为材料,通过筛选DNA聚合酶的种类、改变黄瓜叶片取样量以及扩增产物长度等条件,对叶片直接PCR的体系进行了建立和验证。结果表明,普通Taq酶和Hiproof高保真酶对叶片直接PCR扩增的效果较好,与对照CTAB法提取的DNA相比,20μL、200μL和1 000μL枪头的叶片取样量、不同GC含量(25%~65%)和不同扩增长度的DNA模板对PCR扩增效果无明显差异;此体系也可用于黄瓜的茎、花、果实等组织部位上。该试验方法的建立及应用,省去了DNA体外提取过程,有助于提高基于黄瓜PCR基础上的分子检测效率。     

高通量的PCR模板植物基因组DNA制备方法

制备大量生物样品的模板DNA用于PCR检测是费时费人工的工作。本文介绍一种高通量的植物基因组DNA (gDNA)快速制备及其用于PCR基因型检测的操作方法。将一小段单子叶植物苗叶片(长度约30 mm或40 mm,与96方孔板的孔深大致相同) 或一小块(约2~4 mg)双子叶植物叶片放入96方孔板的各孔中、放入一粒钨合金珠和150 µL制备缓冲液,盖好硅橡胶盖,在涡旋器振动3~5 min破碎组织。用96针复制器(或多通道移液器)转移每样品约0.5~1.0 µL此粗制gDNA溶液(含有2~3 ng gDNA µL^-1)到96孔PCR板的反应液中,适合用各种类型的PCR标记(如简单序列重复SSR,插入缺失InDel等)进行基因型检测,或较大的DNA片段(>1 kb)的扩增,可以得到良好的效果。本方法的关键是控制合适的破碎叶片量与制备溶液量比例(约2~5 mg, 但不超过10 mg 150 µL^-1溶液),以及不要加入过多量的gDNA (不超过PCR反应液量的1/10),以免带入过量的杂质抑制PCR。因此,这种从种植材料,制备gDNA, 转移样品gDNA,到PCR都是96格式化操作的高通量、低成本方法适合于大量植物样品的规模化的基因型检测。     

一种应用PCR缓冲液快速制备水稻DNA模板的方法

本研究介绍一种应用PCR缓冲液快速制备水稻DNA模板的方法。操作方法如下:取少量水稻叶片,用剪刀剪碎后移至200μL离心管或96孔PCR板中,在每个样品中加入50μL 0.5倍的PCR扩增缓冲液,置于PCR基因扩增仪上在95℃条件下煮12 min,取出,无需离心,所得提取液直接作为DNA模板用于PCR扩增。结果表明,该方法所制备的水稻DNA模板在PCR扩增中稳定、准确,其效果与用常规SDS法提取的相当,且这种DNA模板可在短期内保存而不影响扩增效率。该方法具有成本低、操作快速简便等优点,适合推广应用。     

苹果不同HRM反应体系分析效果评价

高分辨率熔解曲线(high resolution melting,HRM)分析是一种新型的DNA多态性快速筛查技术。本研究以苹果品种"富士"和"舞姿"为试材,用来自苹果基因组的SSR标记CH03d11对不同反应体积、不同DNA浓度以及不同PCR退火程序下的HRM分型效果进行了测试与评估。结果表明:采用5μL反应体积可以获得与10μL或20μL反应体积相同的检测效果。在5μL反应体积中,DNA浓度小到0.25ng/μL时,PCR扩增及随后的HRM分析效果依然良好。另外,研究表明,采用降落PCR扩增模式也能取得良好的HRM多态性检测结果。     

巢式PCR和SYBR Green I实时PCR检测转基因大豆方法的研究

为了建立转基因大豆检测技术,采用巢式PCR和SYBR Green I实时PCR技术,检测转基因大豆 外源基因(CaMV35S、CP4EPSPS).结果表明,利用巢式PCR可检测出1 ng/μL转基因含量1％　的大豆中　的CaMV35S基因,而轮PCR的检测限为100 ng/μL;利用SYBR Green I染料能结合双链DNA的 特点,应用实时PCR技术可检测到CaMV35S、CP4EPSPS基因扩增所产生的信号,通过扩增产物的熔解 曲线能有效地区分特异性产物,CaMV35S基因的检测限为0.1 ng/μL.同时利用该方法对黄豆、炒黄豆、豆干等样品进行检测,样品中未检出CaMV35S基因成分.巢式PCR方法明显提高了PCR的检测限, SYBR Green I实时荧光PCR方法能有效、快速检测CaMV35S转基因成分.     

柑桔SRAP和ISSR分子标记技术体系的建立与优化

通过对PCR反应程序、反应体系(DNA模板量、PCR反应体积、Mg2 浓度、dNTP浓度、Taq酶用量、引物量)、电泳检测方法的系统优化,建立了柑桔SRAP-PCR和ISSR-PCR体系;以此进行大规模引物筛选,从而建立了柑桔SRAP和ISSR分子标记技术体系.SRAP-PCR:25μL体系,模板DNA25ng,Tris-HCl10 mmol/L,KCl50 mmol/L,Mg2 1.2 mmol/L,dNTP 120 μmol/L,Taq酶1.5U,引物0.4μmol/L,反应程序为94℃预变性5min,35个循环(94℃ 30s,47℃ 1min,72℃ 1min),72℃延伸10min;ISSR-PCR:25μL体系,模板DNA25ng,Tris-HCl10mmol/L,KCl50mmol/L,Mg2 1.6 mmol/L,dNTP200μmol/L,Taq酶1 U,引物0.8μmol/L.筛选出稳定性好、多态性高的24对SRAP引物和13条ISSR引物.     

半巢式反转录实时荧光PCR检测马铃薯黑环斑病毒

本研究根据基因组中RNA依赖的RNA聚合酶(RDRP)保守序列,设计合成了3条巢式PCR引物和1条TaqMAN荧光探针,建立了半巢式反转录实时荧光PCR检测PBRSV的新方法.该方法采用E.Z.N.A TM快速提取植物总RNA,并有机地结合了巢式PCR和TaqMAN探针检测技术.第二步半巢式反转录实时荧光PCR既是对第一步信号的进一步放大,也是对第一步PCR产物的确认,因此,检测的准确性、灵敏度比巢式PCR、单重Realtime PCR等方法高.实验结果表明,该方法检测灵敏度可达300fg/μL植物总RNA.     

小反刍兽疫病毒实时荧光定量PCR检测方法的建立

为了建立一种快速检测小反刍兽疫病毒的方法。根据Gen Bank中公布的PPRV Nigeria75/1株H基因序列设计2对引物,PCR扩增出1 830 bpH基因,构建标准质粒p MD-18-H。以标准质粒优化反应条件,建立了小反刍兽疫病毒H基因SYBR GreenⅠ实时荧光定量PCR检测方法。最优反应体系25μL:2×SYBR qPCR Mix 12. 5μL,模板DNA 1μL,特异引物各0. 2μL(0. 2μmol/L),DEPC H2O 10. 5μL;最佳反应条件:94℃2 min; 94℃5 s,57℃30 s,40个循环,Ct值与标准质粒模板浓度在3. 28×104～3. 28×10-2copies/μL 7个浓度梯度呈良好线性关系y=-2. 913x+36. 904,斜率为-2. 913,扩增效率120. 5%,相关系数R2=0. 994。建立的实时荧光定量PCR检测方法比普通PCR灵敏度高10 000倍,稳定性好,特异性强,对PPRV的准确诊断和定量检测具有重要意义。     

A rapid method for screening barley for aluminium tolerance

Summary A method for rapid screening of large numbers of barley genotypes for aluminium tolerance is described, and the results of a small scale screening of composite cross CC XXX-C given.     

农业植物品种权申请前的准备

一项能够取得品种权的植物新品种,必须同时具备以下几个条件:属于国家植物新品种保护名录范围;不违反国家法律、妨害公共利益或者破坏生态环境;必须具备新颖性、特异性、一致性和稳定性;应当具备适当的品种名称.另外,品种权是不能自动取得的,即使合乎授权条件的植物新品种,也必须履行<中华人民共和国植物新品种保护条例>(以下简称<条例>)和<中华人民共和国植物新品种保护条例实施细则>(以下简称<实施细则>)所规定的品种权申请程序.为了减少品种权申请的盲目性,节省品种权申请人和植物新品种保护办公室双方的人力、物力和财力,品种权申请人在提出申请以前一定要做好以下准备工作.     

Nitrogen response models for winter cereals grown for forage

Forage double cropping can increase production, reduce erosion risk and improve soil health. Farmer experience in the north‐eastern USA shows that winter cereals can, in 3–4 weeks (Feekes 9 harvest), produce high quality forage given sufficient N at dormancy break. Here, we evaluate crop response models to determine the most economic rate of N (MERN) for forage winter cereals. Sixty‐three on‐farm N‐rate trials (0, 34, 67, 101, 135 kg N/ha) were conducted in New York from 2013 to 2016. Trials were divided into four categories: (a) no yield response to N (group 1; 20 trials); (b) yield plateau exceeded the highest N rate (group 2; one trial); (c) the MERN was below the lowest N rate (group 3; seven trials) and (d) all other N‐responsive trials (group 4; 35 trials). For group 4, three statistical models were compared (quadratic plateau, exponential and square root plateau). Statistical, environmental and economic criteria showed that the quadratic plateau fits the data best and had the most stable predictions across scenarios. The four‐category analysis is effective in determining MERNs of individual trials, and the quadratic plateau is best for determining forage winter cereal MERNs and yields at MERNs for individual trials in the north‐eastern USA.     

Contracting for Migration for the Reason of Zoological Environment

Migration for the reason of zoological environment is the measure for solving the crisis of zoological environment. Nowadays, governments play the chief role in the Migration for the reason of zoological environment, but the migration themselves do not have the freedom to decide whether migrate or not. Because the activity of migration is related to the restraint of economy, culture, law, etc. It is the object of migration that protect the zoological environment and improve the welfare of habitant, which must be concerned when constructing the systems of migration. This paper proves the possibility of contracting for migration based on the methodology of the cost-income analysis. On the other hand, it also argues for changing the mode of migration from that controlled by governments to being decided by habitant themselves.     

Method for screening bread wheat for tolerance to boron

A new method for screening wheat for boron tolerance has been developed to overcome the problems of methods used in the past. Seedlings were grown for 10 days while suspended over B-rich solutions. Their root lengths were then measured as an index of tolerance. Five varieties of wheat were screened at seven concentrations of B. Results were obtained more quickly and easily than from the alternative methodologies and compared favourably. Screening of 128 doubled haploid lines from a cross between Halberd and (Wl*MMC) suggested that transgressive segregation had occurred. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genotype-environment relationships for repening time in blackberries and prospects for breeding an early ripening cultivar for Scotland

Summary When grown in Scotland, eight blackberry cultivars of diverse origin showed considerable variation in the dates when their first fruits ripened. These dates were determined in part by variation in the date of their first open flower and in part by variation in the number of days required from flowering to ripening. Genotype x environment interactions were important for both components, but especially for the second, where they could be attributed to heterogeneity of regressions against the environmental means. Temperature was considered the important environmental factor determining the number of days required from flowering to repening, and accumulated heat units successfully quantified the differences between cultivars for this component: cultivars required similar numbers of heat units in each season and there were no interaction effects. Heat units were not useful for study of flowering dates, however.The implications for breeding early ripening cultivars for Scotland are discussed.     

A General Method for the Initial Values for Two-Sided Iteration

In the study on finding a group of inital values for solving operator equation x=G(x)by the two-sided iterative method,some stronger conditions forG(x)have been demandedand some new parameters have to be introduced since 1978,But,as it will be proved in this paper,Itis unnecessary to use any new parameters and other stronger conditions to get a group of initial val-ues of two-sided iterative, as those initial values can be obtained by means of an auxiliary iterativebeginning with operator G(x)itself. The numerical example in this paper illustraves that the methodproposed in this paper is a general method.     

A Composite Index for Statistical Inference for Ranking Metropolitan Areas

ABSTRACT The construction of a composite index is described to rank U.S. metropolitan areas for educational attainment. The suggested methodology departs from traditional rank sum methods in that this approach utilizes data resulting in a continuous scale, whereas in the rank sum method, no use is made of the actual values of the data, but merely their rankings. The proposed index circumvents several practical problems because the numbers generated are metric measures allowing the use of arithmetic and statistical operations. Data used are pupil/teacher ratio, an effort index, and academic options in higher education. Comparisons are made across areas by population size and by geographic region.     

玉米制种缺素症（营养障碍）的防治

1　症状1.1　缺氮 :幼苗矮化 ,瘦弱 ,叶丛黄绿。生长株从叶尖开始变黄 ,沿叶片中脉发展 ,叶片上形成一个“V”形黄化部分 ,致全株黄化 ,后下部叶尖枯死且边缘呈黄绿色。缺氮严重或关键期缺氮 ,果穗小 ,顶部籽粒不充实 ,蛋白质含量低。1.2　缺磷 :嫩株敏感 ,植株矮化 ,叶尖、叶缘失绿呈紫红色 ,后叶端枯死或变成暗紫褐色。根系发达 ,雌穗受粉受阻 ,籽粒不充实 ,果穗少或歪曲。1.3　缺钾 :下部叶片的叶尖、叶缘呈黄色或似火红焦枯 ,后期植株易倒状 ,果穗小 ,顶部发育不良。1.4　缺钙 :植株矮 ,新叶生长受阻 ,新叶尖端几乎完全失绿 ,分泌透明…