RAPD标记在韦塔桉种源遗传结构上的应用

采用适合韦塔桉RAPD分析的PCR扩增体系,对13个不同种源的韦塔桉运用20个不同随机引物进行了DNA序列多态性分析。实验结果表明韦塔桉不同种源间遗传多态性水平较高,共扩增出165个位点,其中多态位点占77.0%;13个种源间遗传分化指数值为0.5717-0.6670,遗传分化百分比值为0.090%-5.989%。根据遗传分化百分比值构建了13个种源之间的聚类图,聚类图显示了韦塔桉种源间的亲缘关系,17830、17831、17832、17833、17834、17835、17836、17837这7个种源相对聚在一起,17838种源与其它种源的相似性水平最低。对韦塔桉不同种源的RAPD研究为其种源的早期选择了科学依据。     

猪链球菌RAPD分析反应条件优化的研究

为了建立猪链球菌的基因分型方法,对影响猪链球菌RAPD分析的两个主要因素进行了反应条件的优化,结果在25μl反应体系中DNA模板的添加量为50ng,Mg^2 的添加量为2.5mmol/L时,可获得理想的扩增结果,并筛选出多态性好且稳定的随机引物,为猪链球菌的基因分型方法的建立奠定了基础。     

盐藻RAPD反应条件的优化

从盐藻中提取基因组DNA作为模板进行RAPD反应的优化试验 ,获得PCR扩增反应的最佳条件 :随机引物为CPS32 0 (5′ GGCTCATGTG 3′) ;2 0 μl体系中Mg2 2 .0mmol/L ,退火温度 35℃ ;Taq酶活性值 0 .8U。     

杜鹃花RAPD条件的优化

以井冈山杜鹃为材料建立了杜鹃花RAPD反应优化体系 ,用于杜鹃花遗传多样性分析 ,以改进的CTAB法提取杜鹃花属植物杜鹃花叶片总DNA ,分别测试了镁离子浓度、dNTP浓度、模板DNA含量、引物浓度、DNA聚合酶量对反应结果的影响。通过各因子的组合比较 ,建立了杜鹃花RAPD优化体系 :2 0 μLPCR反应体积 ,10×Taq酶配套缓冲液 (2 μL) ;1UTaq酶 ;模板DNA10ng ;13.32pmol引物 ;2 .12mmol·L- 1 MgCl2 ;dATP、dCTP、dGTP和dTTP各 0 .15mmol·L- 1 。     

甜叶菊RAPD反应体系的优化

以甜叶菊为试材,对影响其RAPD反应体系的7个因子进行优化.结果表明,20 μL的优化体系包括:双蒸水13.6 μL,10×Buffer(含15 mmol/L MgCI2)溶液3μL,2.5 mmol/L的dNTPS 1.2 μL,10 μmol/L的引物1μL,20 ng的模板DNA 1μL,1UTaq聚合酶;热循环...     

药用植物草珊瑚RAPD扩增条件优化

采用CTAB-DNA提取方法,从草珊瑚植物的嫩叶中提取总DNA。以此DNA为模板,优化了草珊瑚RAPD-PCR的反应条件。结果表明,PCR扩增体系最适宜的条件为:反应体积25μL,内含2.5mmol/L Mg2+、1.0UDNA聚合酶、0.4μmol/L引物、60ng模板DNA和0.16mmol/L dNTP。扩增程序为:94℃预变性2min;94℃变性30s,37℃复性30s,72℃延伸80s,40个循环;72℃延伸10min;4℃保存10min。     

白色念珠菌RAPD反应体系优化的研究

建立白色念珠菌RAPD的最佳反应体系,并应用于其基因组DNA扩增。通过单因子试验分别研究了Mg^2＋、dNTPs、Taq酶、引物和模板DNA等浓度对RAPD反应的影响;同时,应用L16（4^5）正交试验研究了DNA模板、Mg^2＋、Taq酶、dNTPs和引物浓度对RAPD反应的影响。以条带稳定、丰富、清晰为标准,获得了白色念珠菌基因组DNA的RAPD扩增优化条件;对于白色念珠菌的最适RAPD反应体系为Mg^2＋1．5mmol／L、dNTPs250μmol／L、引物0．6μmol／L、模板100ng／25μL、TaqDNA聚合酶1．5U／25μL。     

桔梗RAPD反应体系的优化

以通化桔梗为材料,用改进的CTAB法提取桔梗叶片的总DNA,通过对不同镁离子浓度、dNTP浓度、模板DNA含量、引物浓度、DNA聚合酶量条件下的RAPD扩增反应的效果,建立了一个适合桔梗的比较稳定的RAPD反应体系,用于桔梗遗传多样性分析。结果表明,桔梗RAPD扩增反应的最佳体系为:模板DNA20ng,dNTP150μmol/L,引物0.3μmol/L,Mg2+浓度2.0mmol/L,TaqDNA聚合酶1Unit,10×Buff-er2.0μL,PCR反应总体积为20μL。按此优化RAPD条件进行实验,重现性良好。     

白色念珠菌菌丝相培养条件及RAPD反应体系优化的研究

目的优化门色念珠菌菌丝相培养条件,为白色念珠菌菌丝相的分子生物学研究提供必要条件;建立白色念珠菌菌丝相RAPD的最佳反应体系,应用于其基因组DNA扩增。方法在RPM11640培养基的基础上,通过单因素试验研究了小牛血清用量、培养液pH值、培养温度和转种次数等对白色念珠菌菌丝相形成的影响。采用单因素试验,分别研究了Mg^2＋浓度、dNTPs浓度、Taq酶的浓度、引物浓度和模板DNA浓度对白色念珠菌菌丝相RAPD反应的影响;应用L16（4^5）正交试验对RAPD反应条件进行了优化。结果白色念珠菌菌丝相诱导的最佳条件为：每100ml培养液中的小牛血清用量为10ml,培养液pH值为7．5,培养温度为36℃,转种次数为12次。白色念珠菌菌丝相的最适RAPD反应体系为：Mg^2＋ 1．25mmol／L、dNTPs 0．4mmol／L、随机引物0．1μmol／L、TaqDNA聚合酶5U／50μl、模板DNA495ng／50μl。结论通过单因素和正交试验,获得了较适白色念珠菌菌丝相培养条件和其基因组RAPD扩增条件。     

RAPD应用于蕈菌研究中的条件优化探讨

在进行鹅膏菌属蕈菌遗传多样性的ＲＡＰＤ分析时,对ＲＡＰＤ分析过程中的ＤＮＡ提取方法、ＤＮＡ纯度、模板ＤＮＡ和ｄＮＴＰ浓度、循环次数、ＤＮＡ不同来源等影响因素进行了大量的实验探索,结果表明：ＤＮＡ不同提取方法具有相同的扩增产物,ＤＮＡ模板中的ＲＮＡ对扩增产物无影响,模板浓度在一个相当大的范围内（５０～４００μｇ）不影响扩增结果．ｄＮＴＰ浓度达０．７５ｍｍｏｌ／Ｌ时无带谱出现,引物浓度达１μｍｏｌ／Ｌ时出现非特异性带谱,从菌丝体和子实体中提取的ＤＮＡ可获得一致的扩增产物．扩增循环４０～４５周期条件下扩增效果较好,本实验证明了ＲＡＰＤ产物具有很好的重复性,建立了适合蕈菌ＲＡＰＤ分析的ＰＣＲ程序及条件,为ＲＡＰＤ应用于蕈菌的遗传研究打下了良好的基础．     

Moderate-density molecular maps of Eucalyptus urophylla S. T. Blake and E. tereticornis Smith genomes based on RAPD markers

Moderate-density molecular maps were constructed for the genomes of Eucalyptus urophylla S. T. Blake and E. tereticornis Smith using RAPD markers and an interspecific cross between the two species. One hundred and eighty-three primers were employed to generate 245 and 264 parent-specific markers in E. urophylla and E. tereticornis, respectively, as well as 49 parent-shared markers. The normally segregating markers, including 208 (84.9%) specific to maternal E. urophylla, 175 (66.3%) to paternal E. tereticornis, and 48 shared by both parents, were used for framework map construction for each parental species. For maternal E. urophylla, the linkage map consisted of 23 linkage groups, 160 framework markers, and 60 accessory markers, defining a total map distance of 1504.6 cM and an average interval of 11.0 ± 8.07 cM. For paternal E. tereticornis, the linkage map contained 23 linkage groups, 126 framework markers, and 92 accessory markers, defining a total map distance of 1035.7 cM and an average interval of 10.1 ± 7.23 cM. Genome length was estimated at 1585.7 and 1507.5 cM for E. urophylla and E. tereticornis, respectively, indicating map coverage of 94.9 and 68.7% of the corresponding genomes. Construction of such maps will be valuable for quantitative trait loci (QTLs) detection, marker-assisted selection (MAS), comparative mapping, and whole genome based fingerprint characterization in Eucalyptus breeding programs.     

Estimation of outcrossing rate in a breeding population of Eucalyptus urophylla with dominant RAPD and AFLP markers

 Eucalyptus breeding is typically conducted by selection in open-pollinated progenies. As mating is controlled only on the female side of the cross, knowledge of outcrossing versus selfing rates is essential for maintaining adequate levels of genetic variability for continuous gains. Outcrossing rate in an open-pollinated breeding population of Eucalyptus urophylla was estimated by two PCR-based dominant marker technologies, RAPD and AFLP, using 11 open-pollinated progeny arrays of 24 individuals. Estimated outcrossing rates indicate predominant outcrossing and suggest maintenance of adequate genetic variability within families. The multilcous outcrossing rate (t_m) estimated from RAPD markers (0.93±0.027), although in the same range, was higher (α>0.01) than the estimate based on AFLP (0.89±0.033). Both estimates were of similar magnitude to those estimated for natural populations using isozymes. The estimated Wright’s fixation index was lower than expected based on t_m possibly resulting from selection against selfed seedlings when sampling plants for the study. An empirical analysis suggests that 18 is the minimum number of dominant marker loci necessary to achieve robust estimates of t_m. This study demonstrates the usefulness of dominant markers, both RAPD and AFLP, for estimating the outcrossing rate in breeding and natural populations of forest trees. We anticipate an increasing use of such PCR-based technologies in mating-system studies, in view of their high throughput and universality of the reagents, particularly for species where isozyme systems have not yet been optimized. Received: 25 March 1997 / Accepted: 13 May 1997     

Structure of the specific combining ability between two species of Eucalyptus.I. RAPD data

 Within the context of the reciprocal recurrent selection scheme developed in 1989 by CIRAD-Forêt on Eucalyptus, RAPD essays were performed to assess the genetic diversity in the two species E. urophylla and E. grandis. The molecular markers were split into two parts: the specific markers (present with different frequencies in the two species) and the common markers (present with similar frequencies in the two species). The study analyses the structure of genetic diversity within and between the two species of Eucalyptus. Different genetic distances are worked out for use in prediction equations of the individual tree trunk volume of hybrids at 38 months. Each distance is expressed as the sum of the general genetic distance and the specific genetic distance. The general genetic distance based on the double presence plus the double absence of bands seems to be an interesting co-variate to use in a factor regression model. Through this model the distance calculated between species explains the general combining ability (GCA) and the specific combining ability (SCA) of the phenotypic character with a global coefficient of determination of 81.6%. Received: 3 November 1996/Accepted: 8 November 1996     

南丰蜜桔基因组DNA RAPD—PCR最佳反应体系的建立

以南丰蜜桔树叶为基因组DNA提取材料,建立了其RAPD-PCR分析的最佳反应体系模板DNA1.5ng/μl,随机引物0.6μM     

卡瓦胡椒RAPD反应体系的建立

卡瓦胡椒RAPD分子标记的研究,目前国内外尚未见有报道。该试验通过CTAB法提取卡瓦胡椒基因组DNA,通过对模板DNA用量、Mg^2＋浓度、退火温度、电泳上样量等几个单因子试验来建立RAPD稳定扩增体系和反应条件,RAPD扩增结果重复性好,稳定可靠,为卡瓦胡椒RAPD分子标记的研究打下基础。     

Regeneration and transformation of Eucalyptus camaldulensis

Reliable regeneration protocols for Eucalyptus camaldulensis using leaf explants from in vitro-grown plants have been developed. Out of the 24 clones tested 13 were regenerated and of these, 6 showed regeneration from more than 60% of the explants. Identical protocols were also successful in the regeneration of some clones of E. microtheca, E. ochrophloia, E. grandis and E. marginata, but at lower frequencies. Co-cultivation of E. camaldulensis leaf explants with Agrobacterium tumefaciens strains carrying a kanamycin resistance gene and the reporter gene β-glucuronidase (GUS), followed by selection on kanamycin at 9 mg l^–1, allowed the selection of transformed shoots that could be rooted on selective media. Transformation of the plants was verified by staining for the GUS enzyme in various plant tissues, NptII assays and by Southern blotting on isolated DNA using specific probes for both the GUS and selectable marker genes. Transformed tissue was obtained with 5 clones of E. camaldulensis tested and a number of A. tumefaciens strains. However, only 1 clone regenerated transformed whole plants reliably. Received: 14 October 1996 / Revision received: 18 February 1997 / Accepted: 1 April 1997     

Occurrence of arbuscular mycorrhiza on aged Eucalyptus

Eucalyptus despite the fact that Eucalyptus seedlings do form both endomycorrhiza and ectomycorrhiza early during their developement. In the present study, all the structures of arbuscular mycorrhiza were observed within roots of four Eucalyptus species of 15, 17 and more than 50 years old at three different sites in northern Algeria. Arbuscular mycorrhiza frequency was assessed in roots of 15-years old Eucalyptus camaldulensis species, during two periods in 2 consecutive years (July and November of 1996 and 1997). Intensity of root colonization was dependent on the time of sampling and attained 42% in July 1997. Accepted: 17 September 1999     

应用电导率探讨邓恩桉不同优株的抗寒性

本文通过测定邓恩桉不同优株在常温下电导率日变化与不同低温胁迫处理电导率的变化,探讨不同处理条件下,电导率变化与个体抗寒性的关系。结果表明,不同个体在常温下电导率存在日波动变化,但日波动性与抗寒性没有直接联系。在不同低温胁迫处理下, 不同优良个体电导率随抗寒能力不同而变化,抗寒能力越强,电导率发生骤变的低温区间越滞后,且变化幅度也较小。由不同处理下的电导率,分别求得不同个体的logistic方程, 经回归分析得出各不同个体的半致死温度,定量评价不同个体抗寒能力,为抗寒-速生优株筛选提供科学依据。