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。     

红椿SRAP反应体系优化及引物筛选

[目的]优化相关序列扩增多态性(SRAP)体系内的不同组分,建立适用于红椿SRAP分子标记的反应体系,并进一步从SRAP引物组合中筛选出稳定、多态性好的引物组合,为红椿遗传多样性研究奠定试验基础。[方法]针对SRAP-PCR反应体系中5个因素各设置8个水平,先利用单因素试验确定浓度梯度,后在确定的梯度范围内选定4个水平,按照正交试验L16(45)进行优化,结合正交直观分析法和新复极差法对各因素进行优化筛选。[结果]确定最优体系为总体系25μL,模板DNA 25 ng,上下游引物各0.3μmol·L~(-1),Taq DNA聚合酶1U,Mg2+2.5 mmol·L~(-1),d NTP 0.3 mmol·L~(-1)。利用稳定的SRAP-PCR体系,从1 505对SRAP引物组合中筛选出30对优质引物组合。[结论]通过不同种源红椿基因组DNA的重复验证,获得了稳定清晰、多态性较强的扩增条带,表明所确定的最优体系稳定可靠,适用性较强,可用于不同种源红椿遗传多样性研究的后续实验。     

火炬松SSR-PCR反应体系的建立及引物筛选

以火炬松(Pinus teada)幼嫩针叶为材料,采用单因素试验设计分析各组分对SSR-PCR扩增结果的影响,并进一步应用正交试验设计,从DNA模板、引物、d NTPs、Mg2+和Taq酶5个因素3个水平对SSR扩增效果进行研究,确定火炬松SSR-PCR的最佳反应体系。结果表明:各因素不同水平浓度对SSR-PCR反应结果均有影响,火炬松SSR-PCR优化后的反应体系为:DNA模板80 ng、引物0.3μmol/L、dNTPs 0.20 mmol/L、Mg~(2+)2.0 mmol/L、Taq酶1.00 U、1×PCR Buffer,总体积25μL。运用该体系从60对引物中筛选出扩增条带清晰、多态性丰富的SSR引物8对,并用不同火炬松家系进行了检验,证明该体系稳定可靠。     

油料植物光皮树SSR-PCR反应体系优化及引物筛选

建立稳定可靠的SSR-PCR反应体系、筛选具有多态性SSR引物,是木本油料植物光皮树分子标记辅助育种与良种选育的重要基础研究。为给SSR标记技术在光皮树遗传图谱构建、亲缘关系鉴定及分子育种等方面的应用研究奠定基础,利用"Cf-05"正反向引物,采用L16(45)正交试验设计和单因素试验相结合的研究方法,对影响光皮树SSR-PCR反应体系的主要因素(模板DNA、引物、d NTPs、Mg~(2+)、Taq聚合酶浓度及退火温度)进行了试验分析。试验结果表明,光皮树SSR-PCR总体积为15μL的最佳反应体系为:模板DNA用量37.5 ng,正反向引物浓度均为0.6μmol·L~(-1),d NTPs浓度为0.3 mmol·L~(-1),Mg~(2+)的浓度为1.5 mmol·L~(-1),Taq聚合酶用量为1.5 U,最佳退火温度为50℃。运用优化了的光皮树SSR-PCR反应体系,可从71对引物中筛选获得11对能适用于光皮树的SSR引物。研究结果表明,试验所建立的反应体系可进一步用于光皮树SSR遗传多样性分析、遗传图谱构建和分子标记辅助育种等方面的研究中。     

刨花润楠SSR-PCR体系优化及天然种群遗传多样性研究

[目的]建立刨花润楠SSR-PCR最佳反应体系,从樟科树种SSR引物中筛选多态性高的引物,并对刨花润楠遗传多样性进行分析。[方法]对影响PCR反应体系的5个因素设置7个水平,利用正交设计L_(16)(4~5)进行优化,确定最佳体系,并用该体系从187对候选引物中进行引物筛选。利用软件POPGENE1.32、PowerMarkerv3.25、FSTAT、GenAlex6.5、Structure2.3和POPTREE分别计算观测等位基因数目(Na)、观察杂合度(Ho)、期望杂合度(He)、多态性信息量(PIC)、基因流(Nm),进行群体遗传结构分析和UPGMA法聚类分析。[结果]刨花润楠最佳体系(20μL)为:Taq酶1.0 U、dNTPs 0.25 mmol·L~(-1)、Mg~(2+) 1.25 mmol·L~(-1)、引物0.5μmol·L~(-1)、模板50 ng。最终筛选出12对具有多态性高的SSR引物。Na、Ho、He和PIC的平均值分别为15.083、0.576、0.751和0.722,表明种群具有较丰富的遗传多样性;Nm平均值为1.500,种群间存在频繁的基因交流;UPGMA将24个种源聚为3类,与Structure分析结果一致。[结论]本研究成功优化了刨花润楠SSR-PCR反应体系,并获得12对适用于刨花润楠的SSR引物,刨花润楠种群遗传多样性丰富,种群间存在频繁的基因交流,24个种源可聚为3类。     

观光木SSR-PCR反应体系优化及引物筛选

在单因素试验基础上,采用正交试验优化影响观光木SSR-PCR扩增的5种主要影响因素,获得最佳反应体系(10μL)为:Mg2+1.0 mmol,d NTP 0.20 mmol,引物0.25μmol,Taq DNA聚合酶0.75 U,模板DNA 60ng。利用优化SSR-PCR扩增体系,从12对引物中筛选出5对多态性高、重复性好的引物。这为进一步开展观光木种群遗传多样性研究奠定了前期实验基础。     

南方红豆杉SSR-PCR扩增体系的建立

为了建立适合于南方红豆杉(Taxus chinensis var.mairei)种群遗传多样性分析的SSR-PCR扩增体系,在单因素试验确定各因素浓度范围的基础上,采用正交试验设计,对影响南方红豆杉SSR-PCR扩增的主要因素浓度进行了筛选。结果表明,南方红豆杉SSR-PCR最佳反应扩增体系为:20μL反应体系中含有1.50mmol/L Mg2++0.2 mmol/L d NTP+0.5U Taq DNA聚合酶+0.3μmol/L引物+75 ng DNA模板。     

兴安落叶松SSR反应体系的建立与优化

采用L16(4^5)正交试验设计,对影响兴安落叶松SSR-PCR反应体系的主要因素(模板DNA、d NTPs、Mg^2+、Taq聚合酶浓度及引物浓度)进行了试验分析,其结果表明:兴安落叶松SSR-PCR体积为20μL的最佳反应体系是模板DNA用量50 ng、正反向引物浓度均为5μmol·L^-1、d NTPs浓度为2.5 mmol·L^-1、Mg^2+的浓度为2.5 mmol·L^-1、Taq聚合酶用量为1.0 U、最佳退火温度为60℃。试验所建立的反应体系可进一步用于兴安落叶松SSR遗传图谱构建、多样性分析、良种选育等方面的研究中。     

紫椴ISSR-PCR反应体系的建立与优化

分别采用单因子试验和正交设计2种方法对影响紫椴ISSR-PCR反应体系的4个因素(Taq酶,Mg2 ,dNTP,引物)在3个水平上进行优化试验.正交设计选用L9(34)方案,采用直观分析法获得影响因素最佳反应水平.单因子试验分别研究各因素对ISSR-PCR反应的影响情况,找出最佳反应水平.2种方法所得影响因素最佳水平存在差异,通过综合比较与分析,最终建立了紫椴ISSR-PCR的最佳反应体系:在20μL的反应体系中,Taq酶1.0U,Mg2 2.0 mmol·L-1,dNTP 0.20 mmol·L-1,引物0.4μmol·L-1,1×PCR buffer,30 ng模板DNA.在此基础上筛选出14个扩增稳定、多态性丰富的ISSR引物,并通过梯度PCR试验,确定引物最佳退火温度.     

西伯利亚杏SRAP-PCR反应体系的优化研究

以西伯利亚杏为试验材料,进行基因组DNA的提取、扩增及电泳试验。通过L16(45)正交试验,确定西伯利亚杏SRAP-PCR优化反应体系(反应体系总体积20μL):Mg2+2.0 mmol·L-1、dNTPs0.25 mmol·L-1、引物浓度1.2μmol·L-1、Taq聚合酶1.0 U、DNA模板20 ng。采用SRAP引物组合ME5/EM10,对优化的SRAP-PCR反应体系进行初步验证,24个西伯利亚杏无性系SRAP分子标记试验共扩增出谱带431条,引物扩增出23条谱带,其中338条为多态性谱带,多态百分率为78.42%。     

Antimicrobial activity of some endemic plants growing in the Eastern Black Sea Region, Turkey

The Eastern Black Sea Region has an extensive flora because of ample rainfall lasting all year. In this study, antimicrobial effects of 74 crude extracts of 22 endemic plants were investigated. Among the 30 active crude extracts, water-insoluble crude extracts from Betula medwediewii, Heracleum platytaenium, Primula longipes, Anthemis cretica ssp. argaea and Centaurea helenioides were the prominent ones with their MIC values.     

Photosynthetic characteristics of Fagus sylvatica and Quercus robur established for stand conversion from Picea abies

Efforts in Europe to convert Norway spruce (Picea abies) plantations to broadleaf or mixed broadleaf-conifer forests could be bolstered by an increased understanding of how artificial regeneration acclimates and functions under a range of Norway spruce stand conditions. We studied foliage characteristics and leaf-level photosynthesis on 7-year-old European beech (Fagus sylvatica) and pedunculate oak (Quercus robur) regeneration established in open patches and shelterwoods of a partially harvested Norway spruce plantation in southwestern Sweden. Both species exhibited morphological plasticity at the leaf level by developing leaf blades in patches with an average mass per unit area (LMA) 54% greater than of those in shelterwoods, and at the plant level by maintaining a leaf area ratio (LAR) in shelterwoods that was 78% greater than in patches. However, we observed interspecific differences in photosynthetic capacity relative to spruce canopy openness. Photosynthetic capacity (A₁₆₀₀, net photosynthesis at a photosynthetic photon flux density of 1600 μmol photons m⁻² s⁻¹) of beech in respect to the canopy gradient was best related to leaf mass, and declined substantially with increasing canopy openness primarily because leaf nitrogen (N) in this species decreased about 0.9 mg g⁻¹ with each 10% rise in canopy openness. In contrast, A₁₆₀₀ of oak showed a weak response to mass-based N, and furthermore the percentage of N remained constant in oak leaf tissues across the canopy gradient. Therefore, oak photosynthetic capacity along the canopy gradient was best related to leaf area, and increased as the spruce canopy thinned primarily because LMA rose 8.6 g m⁻² for each 10% increase in canopy openness. These findings support the premise that spruce stand structure regulates photosynthetic capacity of beech through processes that determine N status of this species; leaf N (mass basis) was greatest under relatively closed spruce canopies where leaves apparently acclimate by enhancing light harvesting mechanisms. Spruce stand structure regulates photosynthetic capacity of oak through processes that control LMA; LMA was greatest under open spruce canopies of high light availability where leaves apparently acclimate by enhancing CO₂ fixation mechanisms.     

Antibacterial activity of plant extracts from Brazilian southeast region

A screening was conducted with 26 plants collected in the Brazilian southeast region, to identify plant extracts with antibacterial properties against Aeromonas hydrophila, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Initially, the agar diffusion method was employed. Then, those extracts presenting activity were submitted to a broth microdilution assay to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). It was observed that 13 of the tested extracts showed antibacterial activity. The best results were obtained with those from Lantana lilacina and Phyllanthus tenellus.     

Predisposition to bark beetle attack by root herbivores and associated pathogens: Roles in forest decline,gap formation,and persistence of endemic bark beetle populations

Bark beetles are largely known for their ability to undergo intermittent population eruptions that transform entire landscapes and pose significant economic hardships. However, most species do not undergo outbreaks, and eruptive species usually exert only minor disturbances. Understanding the dynamics of tree-killing noneruptive species can provide insights into how beetles persist at low densities, and how some spatiotemporal patterns of host predisposition may more likely favor breaching eruptive thresholds than others. Elucidating mechanisms behind low-density populations is challenging, however, due to the requirement of long-term monitoring and high degrees of spatial and temporal covariance. We censused more than 2700 trees annually over 7 years, and at the end of 17 years, in a mature red pine plantation. Trees were measured for the presence of bark beetles and wood borers that breed within the primary stem, root weevils that breed in root collars, and bark beetles that breed in basal stems. We quantify the sequence of events that drive this decline syndrome, with the primary emergent pattern being an interaction between below- and above-ground herbivores and their fungal symbionts. This interaction results in an expanding forest gap, with subsequent colonization by early-successional vegetation. Spatial position strongly affects the likelihood of tree mortality. A red pine is initially very likely to avoid attack by tree-killing Ips beetles, but attack becomes increasingly likely as the belowground complex spreads to neighboring trees and eventually make trees susceptible. This system is largely internally driven, as there are strong gap edge, but not stand-edge, effects. Additional stressors, such as drought, can provide an intermittent source of susceptible trees to Ips beetles, and elevated temperature slightly accentuates this effect. New gaps can arise from such trees as they subsequently become epicenters for the full complex of organisms associated with this decline, but this is not common. As Ips populations rise, there is some element of positive feedback, in that the proportion of killed trees that were not first colonized by root organisms increases. This positive feedback is very weak, however, and we propose the slope between beetle population density and reliance on host stress as a quantitative distinction along a gradient from noneruptive through eruptive species. Almost all trees colonized by Ips were subsequently colonized by wood borers, likely a source of negative feedback. We discuss implications to our overall understanding of cross-scale interactions, between-guild interactions, forest declines, and eruptive thresholds.     

利用三种梢栖真菌生物防治落叶松枯梢病的研究

从落叶松梢栖真菌筛选出对落叶松枯梢病病原菌有抑制作用的菌株,对落叶松枯梢病进行生物防治。将自落叶松梢部分离到的11种梢栖真菌与落叶松枯梢病病原菌进行对峙培养,根据被覆盖程度、抑菌率、拮抗系数和防治预试验的结果,综合评价后发现:粪生粪壳、深绿木霉和球毛壳3种梢栖真菌对落叶松枯梢病有良好的拮抗作用。利用这3个菌株在黑龙江省勃利县通天一林场国富沟进行林间防治试验,结果表明:防治效果为粪生粪壳>代森锰锌400倍液>深绿木霉>球毛壳;深绿木霉和粪生粪壳喷洒浓度50%和球毛壳喷洒浓度75%时,真菌多样性最多,梢栖真菌系统较其他浓度更为稳定,与化学防治相比,既达到了防治目的又具有不污染环境的优势。     

Aromatic plant-derived essential oil: an alternative larvicide for mosquito control

Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella (mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A. aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil.     

Growth and physiological response of six Australian rainforest tree species to a light gradient

An understanding of growth and photosynthetic potential of subtropical rainforest species to variations in light environment can be useful for determining the sequence of species introductions in rainforest restoration projects and mixed species plantations. We examined the growth and physiology of six Australian subtropical rainforest tree species in a greenhouse consisting of three artificial light environments (10%, 30%, and 60% full sunlight). Morphological responses followed the typical sun-shade dichotomy, with early and late secondary species (Elaeocarpus grandis, Flindersia brayleyana, Flindersia schottiana, and Gmelina leichhardtii) displaying higher relative growth rate (RGR) compared to mature stage species (Cryptocarya erythroxylon and Heritiera trifoliolatum). Growth and photosynthetic performance of most species reached a maximum in 30–60% full sunlight. Physiological responses provided limited evidence of a distinct dichotomy between early and late successional species. E. grandis and F. brayleyana, provided a clear representation of early successional species, with marked increase in A_max in high light and an ability to down regulate photosynthetic machinery in low light conditions. The remaining species (F. schottiana, G. leichhardtii, and H. trifoliolatum) were better represented as falling along a shade-tolerant continuum, with limited ability to adjust physiologically to an increase or decrease in light, maintaining similar A_max across all light environments. Results show that most species belong to a shade-tolerant constituency, with an ability to grow and persist across a wide range of light environments. The species offer a wide range of potential planting scenarios and silvicultural options, with ample potential to achieve rapid canopy closure and rainforest restoration goals.     

Forest thinning and subsequent bark beetle-caused mortality in Northeastern California

The Warner Mountains of northeastern California on the Modoc National Forest experienced a high incidence of tree mortality (2001–2007) that was associated with drought and bark beetle (Coleoptera: Curculionidae, Scolytinae) attack. Various silvicultural thinning treatments were implemented prior to this period of tree mortality to reduce stand density and increase residual tree growth and vigor. Our study: (1) compared bark beetle-caused conifer mortality in forested areas thinned from 1985 to 1998 to similar, non-thinned areas and (2) identified site, stand and individual tree characteristics associated with conifer mortality. We sampled ponderosa pine (Pinus ponderosa var ponderosa Dougl. ex Laws.) and Jeffrey pine (Pinus jeffreyi Grev. and Balf.) trees in pre-commercially thinned and non-thinned plantations and ponderosa pine and white fir (Abies concolor var lowiana Gordon) in mixed conifer forests that were commercially thinned, salvage-thinned, and non-thinned. Clusters of five plots (1/50th ha) and four transects (20.1 × 100.6 m) were sampled to estimate stand, site and tree mortality characteristics. A total of 20 pre-commercially thinned and 13 non-thinned plantation plot clusters as well as 20 commercially thinned, 20 salvage-thinned and 20 non-thinned mixed conifer plot clusters were established. Plantation and mixed conifer data were analyzed separately. In ponderosa pine plantations, mountain pine beetle (Dendroctonus ponderosae Hopkins) (MPB) caused greater density of mortality (trees ha⁻¹ killed) in non-thinned (median 16.1 trees ha⁻¹) compared to the pre-commercially thinned (1.2 trees ha⁻¹) stands. Percent mortality (trees ha⁻¹ killed/trees ha⁻¹ host available) was less in the pre-commercially thinned (median 0.5%) compared to the non-thinned (5.0%) plantation stands. In mixed conifer areas, fir engraver beetles (Scolytus ventralis LeConte) (FEN) caused greater density of white fir mortality in non-thinned (least square mean 44.5 trees ha⁻¹) compared to the commercially thinned (23.8 trees ha⁻¹) and salvage-thinned stands (16.4 trees ha⁻¹). Percent mortality did not differ between commercially thinned (least square mean 12.6%), salvage-thinned (11.0%), and non-thinned (13.1%) mixed conifer stands. Thus, FEN-caused mortality occurred in direct proportion to the density of available white fir. In plantations, density of MPB-caused mortality was associated with treatment and tree density of all species. In mixed conifer areas, density of FEN-caused mortality had a positive association with white fir density and a curvilinear association with elevation.     

Growth and production varies between pair-wise mixtures and monoculture plantations in North Viet Nam

The potential benefits of species mixture were investigated using pair-wise comparisons of four timber tree species in northern Viet Nam. Chukrasia tabularis, Canarium album, Michelia mediocris and Eucalyptus urophylla were grown in monocultures and in pair-wise mixtures. The trial was established as a randomized block design with each treatment replicated four times. Volume production gain or loss in mixtures was assessed by calculating a mixture index, which is defined as Relative Yield Total (RYT). At age 48 months, the trial indicated mixed performance with both positive and negative impacts of growing some species in mixtures compared to monocultures. The largest gain was shown in the mixture of a shade-intolerant species, Chukrasia with a more shade-tolerant species, Michelia (47% gain in relative yield), and Eucalyptus with Michelia (45% gain in relative yield) at the first 38 months. The other three mixtures tested, and which were not successful (i.e. had lower relative yields) at 38 months, was the mixture of all shade-intolerant species, including Chukrasia with Canarium, Canarium with Eucalyptus and Chukrasia with Eucalyptus. This suggests that species with different shade tolerances can form complementary pair-wise mixtures, but this changed significantly (P < 0.05) over the following 10 months. At age 48 months the RYT of Chukrasia with Michelia increased by 12%, those of Michelia with Eucalyptus decreased by 20% at 48 months compared to 38 months, suggesting that Eucalyptus should be thinned at around year 5 years. Likewise, the RYT of Michelia with Canarium declined significantly by 23% at 48 months. The RYT of other tested mixtures remained almost unchanged over time.