Conservation Genetics of an Endangered Herb, Hanabusaya asiatica (Campanulaceae)

Abstract: Hanabusaya asiatica (Nakai) Nakai (Campanulaceae), a bee- pollinated, perennial herb, is restricted to the mountainous regions of the eastern-central Korean peninsula. Allozyme analyses for 348 individuals assessed the levels of genetic diversity for five populations. Spatial autocorrelation statistics were also used to examine the spatial distribution of allozyme polymorphisms. The species maintains high levels of allozyme diversity ( H _eS = 0.217) and it exhibits low allozyme differentiation among populations ( G _ST = 0.132) compared with other endemics (mean H _e = 0.096, G _ST = 0.248). There is an apparent pattern of isolation by distance among populations. These results suggest that H. asiatica is at a genetic equilibrium. A considerable deficit in numbers of heterozygotes suggests mating among relatives in populations. At least three populations of H. asiatica should be sampled or conserved to capture or maintain > 99 % of the genetic diversity in the species as a whole. Within local populations, individuals are distributed in a structured, isolation by distance, manner. Approximate genetic patch width in the populations of H. asiatica examined is 5 - 8 m. For conservation purposes, it is suggested that, in general, the sampling of H. asiatica should be conducted at intervals in order to efficiently sample the genetic diversity across an entire population.     

Evolutionary distances and identification of Candida species in clinical isolates by Randomly Amplified Polymorphic DNA (RAPD)

Fast and reliable identification of different species of the genus Candida is important to define adequate therapeutic decisions, because the different species have highly variable susceptibilities to antifungal drugs; azoles and amphothericin B. Accurate statistical records on case history and epidemiological studies also depend on effective identification. To address this problem we established a RAPD method that enabled direct identification of five very common species of Candida. Initially, reference band patterns were established for C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. One of the primers, M2, showed remarkably conserved intra-specific patterns of approximately 10 bands each, ranging in size from 2.0 to 0.1 kb. These patterns were significantly different and species-specific. Few bands were conserved between different species of Candida, which was assumed to be consistent with their phylogenetic relatedness. In addition, band patterns were constant and reproducible and DNA isolated from single colonies yielded sufficient DNA for identification. The reference band patterns were then used, in blind experiments, to identify species of Candida in 50 randomly chosen samples, including clinical isolates and ATCC strains. RAPD results were 100% consistent with results obtained by conventional diagnostic methods and were achieved in one day instead of several days taken by conventional methods. Because ideal identification methods should be consistent with phylogeny and taxonomy we tested whether RAPD could be used to calculate genetic distances. Comparison of RAPD phylogenetic trees with 18S rRNA trees showed significant differences in tree topologies which indicated that RAPD data could not accurately measure the relative distances between different species. Also, computer simulations of RAPD random patterns were used to test whether the observed degree of RAPD band pattern similarities could occur at random. These simulations suggested that the level of inter-specific band pattern similarities observed in our data could be obtained at random, while intra-specific pattern similarities could not. RAPD would be helpful to discriminate between isolates but not to quantitate the differences. We suggest that the inaccurate estimate of genetic distances from RAPD is a general limitation of the technique and not a specific problem of our identification method. Because of the repetitive character of the target sequences, genetic distances calculated from RAPD could be affected by paralogy, namely, recombination and duplication events not parallel with speciation events. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular evidence for the phylogenetic position ofHanabusaya asiatica Nakai (Campanulaceae), an endemic species in Korea

The phylogenetic relationship of the Korean endemic genus,Hanabusaya, to other campanulaceous genera has been controversial since it was described by Nakai in 1911. Three genera of Campanuloideae,Symphyandra, Adenophora, andCampanula, have been considered closely related by various taxonomists on the basis of anther shape, gross morphology, and pollen characters, respectively. We have tested these competing taxonomic hypotheses using the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 12 taxa representing 7 genera of Campanulaceae. The molecular phylogeny indicates strongly thatHanabusaya is more closely allied toAdenophora than toCampanula orSymphyandra. The phylogenetic affinity ofHanabusaya andAdenophora is supported by a 100% bootstrap value and a high decay index (13). The average sequence divergence value (Kimura’s 2-parameter method) betweenHanabusaya and theAdenophora species is 2.58. The value is significantly (about ten times) lower than the ones observed betweenHanabusaya and the species ofCampanula (average of 23.52) and betweenHanabusaya andSymphyandra (24.95). The ITS sequence phylogeny suggests that some morphological characters, such as fused anthers and corolla shape, are homoplastic in the Campanulaceae genera.     

浙江产车前(Plantago asiatica)种群遗传分化的主坐标分析

运用18个有效的10mer-寡核苷酸引物对浙江产车前（Plantago asiatica)的8个种群基因组DNA进行了PCR扩增,共检测到186个位点,其中多态位点125个,占68.70%,应用Jaccard公式计算了8个种群在125个位点上的相似性,以此为基础,应用主坐标排序,作出了8个种群遗传分化的三维排序图,结果表明,8个种群的遗传分化与地理位置、海拔高度有联系。     

48个烟草品种遗传多样性的RAPD分析

利用RAPD分子标记技术对48个烟草品种进行遗传多样性研究。从200个10bp的随机引物用RAPD方法筛选获得28个多态性引物,然后对48份烟草种质资源的基因组DNA进行扩增,共获得184条DNA扩增片断带。其中多态性带86条,平均多态检出率为46.7%。48份材料的遗传距离为0~7.81,采用UPGMA法聚类分析,可将其分为两大类群,即黄花烟草群与普通烟草群,后者又可分为4组。     

烟草RAPD双引物与单引物PCR扩增多态性效率的比较分析

以两个亲缘关系较远的烟草（Nicotiana tabacum）品种台烟7号和白肋21为材料（GS=0.58）,研究了单引物扩增和双引物聚合扩增对RAPD-PCR分子标记多态性的影响。结果显示：双引物反应能够比单引物反应扩增出更多的多态性片段,双引物在白肋21和台烟7号中扩增出的多态性片段总数是单引物反应扩增出的多态性片段总数的6.6倍。因此,双引物RAPD的运用有助于提高烟草多态性分子标记的有效性。     

Identification of hazelnut (Corylus avellana) cultivars by RAPD analysis

The random amplified polymorphic DNA (RAPD) technique offers a useful tool to detect DNA polymorphisms. It can also be used to distinguish different clones and cultivars. We have developed a comprehensive RAPD-based procedure for the routine molecular typing of various plants. Here we report the application of this technique for the correct identification of six hazelnut cultivars (Corylus avellana) widespread in the Campania region (south Italy). The analysed hazelnut cultivars were successfully distinguished by their RAPD fingerprints using the DNA primers U2, U3, U4, U11 and U14. However, in each cultivar we observed very low genetic heterogeneity among the clonal variants. Since this technique is among the simplest and easiest methods used to fingerprint DNA, it could be easily transferred to less sophisticated laboratory infrastructures (e.g. outstations of crop regulatory agencies). Received: 20 December 1997 / Revision received: 6 August 1998 / Accepted: 13 November 1998     

Analysis of single protoplasts and regenerated plants by PCR and RAPD technology

Summary We investigated the use of the polymerase chain reaction (PCR) and the associated random amplification of polymorphic DNA (RAPD) technique in the analysis of DNA and specific genes in plant cells at different stages of regeneration in in vitro cultures. We demonstrate that both procedures can be used to differentiate reproducibly between closely related species as well as to reveal levels of DNA polymorphism in regenerated plants. We also demonstrate that both procedures, using protocols that we have developed, are applicable at all tissue culture stages, from single isolated protoplasts to regenerated plants. Possible explanations for the variation levels detected in regenerated wheat plants are advanced.     

Limitations of the RAPD technique in phylogeny reconstruction in Drosophila

In this study the limitations of the RAPD technique for phylogenetic analysis of very closely related and less related species of Drosophila are examined. In addition, assumptions of positional homology of amplified fragments in different species are examined by cross-hybridization of RAPD fragments. It is demonstrated that in Drosophila the use of RAPD markers is very efficient in identification of species. For assessment of phylogenetic relationships, however, the method is limited to sibling species, and reliable measures for genetic distances cannot be obtained. Hybridization experiments demonstrate that fragments of similar length amplified from different species are not always derived from corresponding loci, and that not all RAPD fragments within the same amplification pattern are independent.     

Primer Design and Optimization for RAPD Analysis of Nepenthes

Primers with higher G+C content produced better random amplified polymorphic DNA (RAPD) profiles in Nepenthes. The occurrence of clustered G's and C's in the center of the primer seemed also to influence the banding patterns. It was also observed that for certain polymerases, the use of different buffers other than that recommended by the manufacturer provided a better amplification profile for Nepenthes.     

烤烟品种资源的RAPD分析

从156个随机引物中筛选出30个引物,对来自国内外的31个烤烟品种进行了遗传多样性和亲缘关系的RAPD分析。在检测的246个位点中,127个位点为多态位点（52％）。聚类分析表明,不同烤烟品种之间存在明显差异,31个品种基本上可分为4大类,品种最多的第一大类（19个）主要由来自美国的Orinoco烤烟选育而成,反映了我国现在推广的烤烟品种遗传基因比较狭窄。研究结果表明,RAPD技术可用于烤烟品种的鉴别和纯度测定。研究为烤烟杂种育种中亲本的选配提供了一定的理论依据。     

鸭梨及其变异类型的RAPD分析

鸭梨为梨属白梨系统优良资源,生产中其变异类型较多。本文首次对鸭梨及其9个鸭梨变异类型进行RAPD分析,并初步筛选出3个多态性引物即S28、S32、S176。研究发现:芽变品种垂枝鸭梨增加了1条特异带(S32-600)。在芽变品种魏县巨鸭梨、甜鸭梨、垂枝鸭梨的扩增产物中均少1条特异带(S28-400)。魏县巨鸭梨扩增产物中缺少2条特异性条带(S176-900和S176-1150)和阎庄自花结实鸭梨缺少1条特异性条带(S176-1150)。可见魏县巨鸭梨、甜鸭梨、垂枝鸭梨与阎庄自花与其他类型能区分开。     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

荔枝DNA提取及RAPD扩增条件优化

为应用RAPD技术开展对荔枝种质资源的分析,以S43(GTCGCCGTCA)为引物,通过试验设计,分别研究了退火温度、模板浓度、引物浓度、dNTP浓度、Taq DNA聚合酶用量对荔枝RAPD-PCR反应的影响。建立并优化了适宜荔枝RAPD分析的扩增体系:20μL的反应体系,30ng的模板DNA度,0.25μmol/LRAPD引物、1.0UTaqDNA聚合酶,0.2μmol/LdNTP为荔枝适宜的RAPD-PCR扩增条件。     

RAPD技术分析不同抗旱性苜蓿品种DNA的多态性

分别从不同抗旱性的紫花苜蓿品种中挑选抗旱性强的、抗旱性中等的和抗旱性弱的品种各三个,提取叶片基因组DNA,相同抗旱性苜蓿品种DNA等量混合构建三个池DNA。采用RAPD技术分析不同抗旱性混合DNA的多态性,并筛选出标记多态性的引物。结果表明：13组260个随机引物经五轮筛选,得到48个引物对不同抗旱性苜蓿品种池DNA扩增结果产生多态性,多态性引物占18．5％。其中5个引物能够稳定标记池DNA多态性,且特异性明显。表明不同抗旱性苜蓿品种之间具有明显的遗传多样性。     

桃不同类群的遗传多样性及其遗传结构的RAPD分析

采用RAPD技术,利用从200个随机引物筛选的22个十碱基引物对桃10个类群180个样品的DNA进行扩增,得到180个位点。通过对所得的数据统计分析,在类群的遗传多样度上表现为:黄肉桃类>蜜桃类>蟠桃类>红叶桃类>硬肉桃类>碧桃类>水蜜桃类>油桃类>寿星桃类>垂枝桃类。在180个位点中检验出多样度范围在0.4以上的位点分布在桃总群体中的有37个,而在桃类群间的则有13个。聚类分析的结果是:桃食用栽培品种的类群聚为一组;其它非食用桃类群各为一组。对桃类群的遗传多样性及其结构分析,为桃的品种资源保存和利用提供分子生物学依据。     

水稻RAPD反应体系的正交优化

以焦旱1号总DNA为材料,首先对影响RAPD-PCR反应的模板DNA、Mg~(2+)、dNTP、引物和Taq DNA聚合酶浓度等因素进行了初步优化,分析了各因素对RAPD-PCR扩增结果的影响.在此基础上对影响RAPD-PCR反应的Mg~(2+)、dNTP、引物和Taq DNA聚合酶浓度等4个主要因素进行正交优化,研究结果表明:在25 μl RAPD-PCR反应体系中,模板DNA 20 ng;Mg~(2+)浓度1.5mmol/L;dNTP的浓度0.2mmol/L;引物量15 pmol;Taq DNA聚合酶1.0 U.在此最佳条件下,利用引物B8对18个北方粳稻品种进行了成功的扩增.     

8个禾本科草坪草品种遗传多样性的RAPD分析

利用RAPD技术,对禾本科4属4种8个品种的草坪草进行遗传多样性分析。15个有效引物用于PCR扩增,共获得RAPD谱带144条带,多态性带占63.2%。8个草坪草品种间的遗传距离在0.0857~0.2928之间,脱壳狗牙根与普通狗牙根间的遗传距离最小,交战2号与爱神特之间的遗传距离最大。用UPGMA和邻接法进行聚类分析,得到2个拓扑结构基本一致的树系图。同一种不同品种间的亲缘关系最近;不同属种间的遗传距离加大,同族不同属的草坪草基本构成一支;4个暖季型草坪草品种构成一个单系类群,但属于冷季型草种的黑麦草(爱神特)单独构成一支,并未与同族的其它3个冷季型草坪草品种(高羊茅)聚在一起。本研究不支持将黑麦草属与羊茅属放在同一族内的分类处理,并建议将高羊茅划分为过渡类型的草坪草。     

卡瓦胡椒及胡椒的RAPD聚类分析

目的：通过RAPD分析搞清楚卡瓦胡椒与胡椒及胡椒属其它近缘野生种的亲缘关系。方法和结果：采用随机引物80条进行筛选,用从80条随机引物中入选的20条引物,对卡瓦胡椒和胡椒属共计28份材料进行RAPD扩增,均能产生清晰的扩增谱带。重复1—2次,结果稳定可靠。20个引物共扩增出170个条带,其中多态性条带有20条,占总扩增条带数的12％。RAPD分析结果显示在相似系数0．36处对28份种质可划分为6个类型,其中卡瓦胡椒被单独聚为一类,说明卡瓦胡椒与胡椒及其它近缘野生种的亲缘关系有一定的距离。     

甘草亲缘关系的RAPD鉴定

以不同地区的3种15组甘草(Glycyrrhiza uralensis、G．infleta、G．glebra)种子为材料,探讨RAPD分子标记在研究、鉴定甘草亲缘关系中的可行性。从50个随机引物中筛选出9个有效引物,9个引物共扩增出961条DNA带,其中多态性条带811条,占总扩增条带的84．4％。对扩增出来的961条DNA带进行分析,结果表明：15组甘草植物的平均遗传距离为0．41,其中采自甘肃民勤野生甘草与采自新疆布尔津乌拉尔野生甘草遗传距离最小,为0．26;而采自内蒙古杭锦旗上海升袖的野生甘草与采自青海贵德的乌拉尔野生甘草遗传距离最大,为0．63。由RAPD聚类分析结果得出15组甘草植物之间的亲缘关系与形态学分类结果存在差异。