红松SSR-PCR反应体系的建立与优化

为了建立红松SSR-PCR最佳反应体系,以CTAB法提取的红松针叶DNA为模板,应用单因子试验及L16(45)正交试验系统分析了DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度、Taq酶浓度等对SSR-PCR扩增结果的影响,并对延伸时间、热循环参数进行了探讨,建立了稳定的、可重复的红松SSR-PCR最佳反应体系及PCR扩增参数。结果表明,PCR的最佳反应体系为:10μL体系中,1×PCRbuffer1μL、DNA模板50～120ng,Mg2+3mmol/L,dNTP0.4mmol/L,引物0.5μmol/L,Taq酶0.75U。利用该体系筛选出扩增条带清晰、多态性丰富的SSR引物13对。在此反应体系下,所扩增谱带清晰、稳定、多态性高。     

漾濞泡核桃ISSR-PCR反应体系的建立和优化

以漾濞泡核桃优株为研究对象,研究其ISSR-PCR反应体系中的5个影响因子(模板DNA浓度、Taq DNA聚合酶浓度、引物浓度、dNTPs浓度、Mg2+浓度)对PCR扩增效果的影响,从而建立起漾濞泡核桃的最佳ISSR-PCR反应体系。结果表明:模板DNA最佳浓度为50 ng/(25μL),Taq聚合酶浓度为0.75U/(25μL),引物浓度为0.7mmol/L,dNTPs浓度为0.20mmol/L,Mg2+浓度为2.0mmol/L,利用该最佳体系对试验中所选取的样本进行扩增反应。建立了适宜于漾濞泡核桃的ISSR-PCR扩增的最佳体系,该体系的建立为利用ISSR分子标记技术对漾濞泡核桃进行种质资源研究奠定了基础。     

江南油杉ISSR-PCR反应体系建立与引物筛选

为了建立江南油杉(Keteleeria fortunei var.cyclolepis)ISSR-PCR最佳反应体系,以江南油杉嫩叶为材料,采用改良CTAB法提取基因组DNA,通过单因素实验设计,进行ISSR-PCR反应体系和反应条件优化,并筛选出多态性ISSR引物。实验结果表明:江南油杉ISSR-PCR最佳反应体系为25μL体系中模板DNA 40 ng,引物浓度为0.8μmol/L,2×Taq PCR Master Mix用量为12μL。利用该体系从100条ISSR引物中筛选出扩增条带清晰、特异性好的6条引物(UBC807、UBC809、UBC811、UBC812、UBC835、UBC841)。     

桑天牛线粒体细胞色素氧化酶Ⅰ基因的PCR反应体系优化

建立一整套适用于研究桑天牛线粒体细胞色素氧化酶I(mtDNA CO I)基因的PCR反应体系,研究不同地理分布的桑天牛遗传多样性。通过L_(16)(4~5)正交组合试验和单因素梯度试验对缓冲液(Mg~(2+))、dNTP、随机引物、TaqDNA聚合酶、模板DNA的浓度和退火温度、循环次数等影响PCR扩增的重要因素进行优化。试验结果表明,50μL反应体系及反应程序中各因素优化组合为:缓冲液(含Mg~(2+))0.9mmol/L,dNTP 0.15mmol/L,随机引物0.44μnol/L,TaqDNA聚合酶3.0U,模板DNA75ng;退火温度47℃,循环次数35次。     

杉木SSR-PCR体系优化

SSR-PCR反应体系优化是杉木SSR标记研究的基础.利用正交L16 (45)设计实验对杉木SSR-PCR反应体系的Mg2+、dNTPs、引物、Taq酶和DNA浓度5个因素的4个水平进行优化试验.并在正交直观分析和方差分析的基础上,分别对Mg2+、dNTPs进行单因素确定.获得的最佳反应体系为:在20 μL反应体系中,Mg2浓度为1.0mmol/L,引物浓度为0.25 μμmol/L,dNTPs浓度为0.15 mmol/L,Taq酶为0.05 U/μL,DNA为30 ng,10×PCR Buffer 2μL,不足部分用双蒸水补齐至20μL.在最佳反应体系的基础上,采用单因素实验确定了引物的最佳退火范围为63～53℃.利用此反应体系对10个F1代杉木材料及其亲本,进行4对SSR标记进行PCR扩增并电泳检测,其结果清晰、稳定.说明此体系适合进一步的杉木SSR研究工作.     

萱草属植物ISSR-PCR反应体系的优化

以萱草属的3种植物为材料,对影响ISSR-PCR扩增效果的因素Taq酶用量、Mg2 浓度、dNTP用量、引物用量、模板DNA浓度以及退火温度等指标进行单因子筛选和优化,建立了适合萱草属植物ISSR-PCR分析的最佳PCR反应体系:20μL反应体系中Taq酶0.6U、Mg2 1.8 mmol/L、1×Buffer 2 μL、dNTP0.2 mmol/L、引物0.6mmol/L、DNA模板10 ng/μL.扩增程序为94℃预变性5 min,94℃变性40 s,52℃退火45 s,72℃延伸1.50 min,共39个循环;72℃完全延伸5 min,4℃保持.该反应体系及扩增程序扩增谱带清晰,产物量多,为利用ISSR-PCR分子标记研究萱草属植物的遗传多样性提供标准反应条件.     

刨花润楠SRAP-PCR体系建立与优化

以刨花润楠(Machilus pauhoi)1.5 a生小苗幼嫩叶片为试材,对影响刨花润楠SRAP-PCR扩增的模板DNA量、引物、dNTP和Mg2+体积摩尔浓度、Taq DNA聚合酶、退火温度6个主要因素进行优化.结果表明,SRAP-PCR的最佳反应体系为:25μL的SRAP-PCR反应体系中,2.5μL 10×PCR buffer、模板DNA量60 ng、Mg2+2.0 mmol/L、dNTP 0.225 mmol/L、引物0.3μmol/L和Taq DNA聚合酶1.25 U.对优化的反应体系和扩增程序的验证结果表明,优化的刨花润楠SRAP-PCR反应体系和扩增程序是稳定可行的.     

杨树基因组SRAP扩增体系的建立与优化

以响叶杨和银白杨及F1代为实验材料,利用正交设计直观分析法和单因素随机试验对杨树SRAP反应体系中各组分(TaqDNA聚合酶、dNTP、引物和Mg2+)的浓度进行优化;通过实验确定10μL反应体系为:引物0.35μmol/L,TaqDNA聚合酶0.5U,dNTP0.2 mmol/L,Mg2+2.0 mmol/L,10×PCRbuffer 1μL,模板DNA 1μl(20 ng/μL).同时利用最佳反应体系以及正交设计直观分析法对PCR扩增程序进行筛选,并通过梯度PCR试验,确定引物最佳退火温度.结果表明最佳反应程序为:开始94℃变性5min,反应前5个循环在94℃1 min,37℃30 s,72℃ 1min条件下运行;之后35个循环在94℃1 min,54℃ 1 min,72℃ 1 min条件下运行,最后延伸7 min;12℃保存.并利用两树种的杂种F1代对优化的反应体系和程序的可行性进行了验证.     

红椿SSR-PCR体系建立和多态性引物筛选

[目的]本研究旨在建立红椿SSR-PCR最佳反应体系,并筛选适于红椿SSR分析的高多态性引物。[方法]通过L16(45)正交试验设计,确立红椿SSR-PCR最佳反应体系;利用优化后的体系对来自楝科植物的135对SSR引物,在6个不同的红椿居群中进行扩增,筛出能有效扩增的引物并进一步筛选出适于红椿的高多态性引物。[结果](1)10μL基于荧光d UTP的SSR-PCR体系中包含:10×buffer 1.0μL,Taq酶(5 U·μL-1)0.1μL,Mg Cl2(25mmol·L-1)0.8μL,d NTP(200 mmol·L-1)0.025μL,荧光d UTP(1 nmol·μL-1)0.01μL,引物(10 mmol·L-1)0.8μL,DNA模板45 ng剩余用dd H2O补足;(2)筛选出29对能有效扩增的引物,复选后获得了12对适于红椿SSR分析的高多态性引物。[结论]建立了SSR-PCR最佳反应体系并筛选出高多态性引物,为红椿的分子标记等遗传学研究提供了基础。     

红豆杉TbAP2基因荧光定量PCR体系的建立及优化

[目的]建立稳定可靠的、适合检测红豆杉(Taxus L.)TbAP2基因表达量的荧光定量PCR实验体系。对于检测该物种中基因的组织特异性表达具有重要意义。[方法]以曼地亚红豆杉细胞为试材,提取总RNA并反转录为cDNA,根据TbAP2基因序列设计多对引物,合成内参基因TBC41的引物,采用正交试验L_9(3~4)方法分别筛选以上2个基因5μL和10μL小反应体系及20μL常用体系中的最佳组合,并通过cDNA模板用量和引物用量等方面进行优化,以确保基因扩增效率在90%～105%之间。[结果]本研究建立了TBC41和TbAP2基因在5、10、20μL体系下的荧光定量最佳PCR反应体系,在优化后的5μL体系下,加入Mix(2×) Universal 2.5μL,cDNA模板1.0μL,正反引物共1.5μL,内参基因TBC41和目的基因TbAP2的扩增效率均为94%;在优化后的10μL下,加入Mix(2×) Universal 5μL,cDNA模板1.2μL,正反引物共1.3μL,TBC41和TbAP2的扩增效率分别为95%和94%。在优化后的20μL下,加入Mix(2×) Universal 10μL,cDNA模板0.5μL,正反引物共1.5μL,TBC41和TbAP2的扩增效率分别为93%和99%,以上各扩增体系回归系数R~2均大于0.980。[结论]在以上3种反应体系下,内参基因和目的基因均具有接近100%的扩增效率,表明本研究成功建立了适合检测红豆杉TbAP2基因表达量的荧光定量PCR实验体系,并为红豆杉其它基因的表达研究提供参考。     

Browsing Damage on Pine (Pinus sylvestris and P. contorta) by a migrating moose (Alces alces) Population in Winter: Relation to Habitat Composition and Road Barriers

In Scandinavia, moose (Alces alces L.) sometimes cause severe browsing damage to economically-important pine. Moose-vehicle accidents have spurred construction of fences along roads, and these may interfere with moose migration between summer and winter ranges, or the road alone may be a barrier. If this happens and moose build up along roads, landowners may suffer economically. Therefore, this study investigated whether roads, fences or other factors influence the use of young pine stands by moose. Eighty stands along roads in northern Sweden were evaluated in which individually-browsed branches were counted on 9972 pines. Moose browsing was not significantly related to birch (Betula pendula Roth, B. pubescens Ehrh.) density, nor did it differ between pines (Pinus contorta Douglas or P. sylvestris L.). However, increased pine density, site productivity and proximity to a highway were associated with increased browsing. Further large-scale studies are needed to understand moose habitat selection and the effects of roads.     

Effects of Root Freezing on the Physiology and Growth of Picea glauca,Picea mariana and Pinus banksiana Seedlings Under Different Soil Moisture Regimes

The root systems of 2-yr-old Picea glauca, Picea mariana and Pinus banksiana seedlings were submitted to various frost temperatures during an artificial frost to induce different levels of root damage. Frost-damaged and control seedlings were placed in a greenhouse under high and low soil moisture regimes. Seedling growth and physiology were evaluated periodically. Seedling survival was reduced when root damage reached levels of 60-80%. Root systems of all three species showed partial to total recovery by the end of the experiment. In general, root freezing damage caused reductions in seedling growth, with these reductions becoming less significant over time. Root damage had little to no effect on black spruce and jack pine seedling physiology, while white spruce CO 2 uptake decreased with increasing root damage. Shoot nitrogen content of all three species decreased slightly with increasing root damage.     

Stand Structure in Pine,Spruce and Deciduous Forests: A Comparison Between Finnish and Russian Karelia

This study investigated the stand structure in pine, spruce and deciduous forests in the border district of Finland and Russia. A total of 46 mature forest stands was selected as pairs, the members of each pair being as similar as possible with respect to their forest site type, age, moisture and topography. The stands were then compared between the two countries by means of basal areas and number of stems. The proportions of dominating tree species were 2-12% lower, and correspondingly the proportions of secondary tree species higher, in Russian forests. The density of the forest stock was also higher in each forest type in Russia. The forests in the two countries differed most radically in terms of the abundance of dead trees. The amount was two to four times higher in Russian deciduous and spruce forests, and in pine forests the difference was 10-fold. The stand structures indicated that Russian coniferous stands, in particular, were more heterogeneous than intensively managed pine and spruce stands in Finland.     

Growth and Terminal Bud Formation in Picea abies Seedlings Grown with Alternating Diurnal Temperature and Different Light Qualities

The root collar diameter and the height:diameter ratio are of particular importance in container-grown seedlings where a high density in the containers may produce spindly seedlings. Temperature regimes and light quality are known to affect plant growth. The aim of this study was to identify responses in Picea abies (L.) Karst. seedlings grown with light providing different red:far-red ratios and under temperature regimes with alternating day (DT) and night temperature (NT) from negative (DT < NT) to positive (DT > NT) difference (DIF) between DT and NT. Experiments were conducted in controlled environment chambers and in a daylight phytotron. Only limited thermoperiodic responses appeared in P . abies seedlings with respect to seedling height and dry weight accumulation. The formation of terminal buds, however, was clearly delayed in seedlings grown at negative DIF. The results indicate a requirement for day extension light that is high in far-red, to prevent terminal bud formation under natural short-day conditions. An extended study should be conducted to clarify the minimum level of light intensity and the optimal light quality needed to prevent terminal bud formation under natural short-day conditions.     

CAD与建筑设计的关系

本文分析了CAD在设计中引起的正面和负面影响,并进行了系统的阐述,从而使设计者在应用中能够保持客观的态度。     

Natural Regeneration in Clear-cuts: Effects of Scarification,Slash Removal and Clear-cut Age

The effects of soil scarification (mounding), slash removal and clear-cut age on the natural regeneration in clear-cuts was evaluated using data from four sites in southern Sweden. The treatments were carried out during a good seed and establishment year for birch ( Betula pubescens Ehrh. and B. pendula Roth). Scarification had the strongest positive effect on the density of naturally regenerated seedlings, especially in birch, but also in pine ( Pinus sylvestris L.) and spruce [ Picea abies (L.) Karst]. Slash removal had a positive effect on birch density. No statistically significant effect of clear-cut age was found. In addition, no statistically significant interactions between clear-cut age and scarification or slash removal were found. The ingrowth of field vegetation was the fastest in areas that were not scarified, less rapid in areas scarified on old clear-cuts, and the slowest after scarification in fresh clear-cuts. In conclusion, it may be possible to control the density of birch during a good establishment year for birch. If birch is desired, the best combination of treatments is to remove the slash and scarify; otherwise, these treatments should be avoided.     

Risk Perception Among Non-industrial Private Forest Owners

Based on an enquiry, risk perception among non-industrial private forest owners is described in relation to climate change and forestry hazards. Of the respondents, 11% took action to remedy the effects of climate change. Out of a given set, hazards were ranked according to each respondent's experience of recent substantial financial loss to the estate and in relation to his or her willingness to make investments aimed at risk reduction. For each hazard, the respondent assessed the risk in four classes ranging from very high to negligible risk. Six hazards were considered most problematic in all three aspects: browsing damage, falling timber prices, damage by wind, spruce bark beetle, root rot and pine weevil. A majority of the respondents claimed to take action to reduce the risk associated with at least one hazard, while 35% did not know whether they did. Excluding climate change, the need for decision support was the largest in relation to damage by wind owing to a combination of perceived high risk and a high level of ignorance in relation to whether risk-reducing measures were taken.     

Interactions in decomposition and N mineralization between tropical legume residue components

In situ produced plant residues contain a mixture of different plant components of varying quality. To assess synergistic or antagonistic interactions occurring during the decomposition and mineralization of such mixtures, individual plant parts (stems, leaves, leaf litter and roots) or the mixture of stems, leaves and leaf litter of the agroforestry species pigeonpea (Cajanus cajan) or of crop residues of peanut (Arachis hypogaea) or of the weed hairy indigo (Indigofera hirsuta) were incubated in pots for 19 weeks. Periodically, remaining plant residues were sieved out (>2 mm), weighed and N content as well as soil mineral N determined. Above- and below-ground residues of peanut decomposed fastest and showed the largest N release in agreement with their high N concentration and low-acid detergent fibre (ADF) : N ratio. Hairy indigo was hypothesized to be of lower quality than pigeonpea because of its high-polyphenol content. However, it decomposed faster than pigeonpea, largely because of the higher N and lower lignin concentration of its components. Ranking of individual plant components for N mineralization resulted in the following pattern, leaves > leaf litter > roots > stems. In mixtures of the different plant components a similar species order in decomposition was obtained, e.g. peanut > hairy indigo > pigeonpea. The amount of N released from the mixture was dominated by stem material that comprised 46–61% of the mixture. The interactions in mixtures were relatively small for peanut (generally high-quality components) as well as for pigeonpea (low proportion of high-quality components, i.e. N rich leaf material). However, a positive interaction occurred during later stages of N mineralization in the mixture of hairy indigo as it had a significant proportion of N rich components and absence of highly reactive polyphenols. Thus, for plants with low to intermediate chemical quality attributes, manipulating plant composition (e.g. by varying harvest age, affecting stem and leaf proportions) will be important to obtain significant interactions during decomposition when its components are mixed.     

Pasture production and tree growth in a young pine plantation fertilized with inorganic fertilizers and milk sewage in northwestern Spain

The use of organic waste materials such as milk sewage as an organic fertilizer could have the dual advantages of organic-waste disposal and reduced dependence on inorganic fertilizers. The effects of fertilization with (1) conventional mineral fertilization, (2) milk sewage sludge at 40 kg N ha⁻¹ target rate and (3) no fertilization on pasture production and tree growth were examined in an experiment consisting of two pasture mixtures under a one-year-old Pinus radiata plantation with a density of 2500 trees ha⁻¹. The two pasture mixtures were: (1) Dactylis glomerata L. var. saborto (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹); (2) Lolium perenne L. var. Tove (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹). The experiment began in the spring of 1995 using a randomized block design with three replicates in Castro Riberas de Lea (Lugo, Galicia, north-western Spain). Plot size was 12 × 8 m², with a 1 m buffer strip between plots. Two-year data showed that fertilization with either material had a positive effect on pasture production, with no significant difference between the two fertilization treatments. Tree growth in the milk sewage sludge plot was significantly higher than in the control plots. Inorganic fertilization increased pasture production, but affected tree growth negatively. The results show that milk sewage sludge could be used as a fertilizer in silvo-pastoral systems. This revised version was published online in June 2006 with corrections to the Cover Date.