用RAPD技术检测不同生态类型蜘蛛代表品种的基因组DNA多态性

蜘蛛有3种生态类型,即洞穴型、结网型和游猎型。利用RAPD技术检测3种生态类型蜘蛛的代表品种,即白额巨蟹蛛(游猎型)、漏斗蛛(结网型)、虎纹捕鸟蛛和七纺器蛛(洞穴型)的基因组DNA的多态性。用11个随机引物对3种生态类型的代表蜘蛛的基因组DNA进行扩增,平均每个品种观察到约22.5个标记,单个引物获得的标记在4～13个之间。实验结果经统计学分析表明,L纺器蛛和虎纹捕鸟蛛的随机扩增多态DNA共享度(F)高,在分子水平上进一步证实了同生态类型蜘蛛的亲缘天系最近,聚类结果表明,3种类型蜘蛛的总体演化趋势为:洞穴型 结网型—游猎型,与以往的形态学研究相符。     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

用RAPD技术对特化等级裂腹鱼类亲缘关系的探讨

为确定特化等级裂腹鱼类的遗传分化关系 ,对 7种 2 2个裂腹鱼个体进行随机扩增多态DNA(RAPD)研究。从所使用的 40个随机引物中 ,选择了 37个扩增带谱清晰的引物进行分析。根据构建的分子系统树显示 ,特化等级裂腹鱼类划分为 3个属级分类单元较为合适 ,这与采用形态学特征进行系统分析所得出的结论一致。在 2 2个个体之间遗传距离矩阵中 ,最大的遗传距离指数达到了 90 38% ,此外 ,还有较多的遗传距离指数也在 6 0 %～ 90 %之间。通过分析 ,认为用RAPD标记技术来分析属级或属级以上分类单元的亲缘关系 ,具有一定的适用性。对于一些遗传差异较大的类群 (如裂腹鱼类 ) ,应用属级或属级以上分类单元的亲缘关系 ,很可能会出现个体之间遗传距离接近或达到 10 0 %的情况 ,从而无法探讨其相互之间的亲缘关系。     

Adaptations of Cave Fishes with some Comparisons to Deep-sea Fishes

I provide my retrospective and prospective views on adaptations of cave fishes. I emphasize the history of my insights into cave adaptation from 45 years of research using surface, cave-spring, and cave species of amblyopsid fishes. My approach has been to use natural experiments and to always consider multiple hypotheses. To clarify evolutionary adaptations, I show the importance of a broad comparative approach which includes studies of morphology, metabolic physiology, foraging behavior, life history, and ecology. And I show that the most important agents of selection, of darkness and attendant low food supply, are best understood in the context of rigor, variability, and predictability. I also present my insights from what I consider the most insightful contributions on deep-sea fishes. The contributions are those of Marshall in studies of morphology in relation to energy economy of pelagic and benthic species, Childress in studies of physiological and biochemical adaptations with depth for pelagic species, and Koslow in studies on population biology and life history of bathybenthic and benthic sea-mount species. Compared to caves, I suggest that the extremes of metabolic and life history adaptations of deep-sea fish are explained by a longer evolutionary history and a much greater habitat range, food supply, and predation risk. Finally, I take a retrospective view of what we have learned about cave fishes. I discuss possible evolutionary mechanisms that can explain the trends with increasing cave adaptation in amblyopsid fishes, especially progenesis and the pleiotropic effects of the stress resistance syndrome. Finally, based on insights from deep-sea fishes, and emerging new techniques, I suggest what cave fish biologists should do in the future.     

应用RAPD分子标记技术探讨3种石斛属植物的种间关系

采用RAPD分子标记技术,分析了金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的种间关系。10个引物产生的113条DNA扩增片段中,106条（93.81%）具有多态性,利用113个RAPD标记,计算遗传距离,利用非加权组平均法建立聚类图。结果表明,RAPD标记技术较好地从分子水平揭示金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的遗传背景、亲缘关系,并为后期在DNA水平上对药用石斛的开发利用提供资料。     

利用RAPD分子标记评价仲彬草属的种间关系

利用随机扩增多态性DNA(RAPD)技术分析了14种仲彬草属Kengyilia植物的种间关系。对34个OPRON公司十聚体随机引物进行多态性筛选,20个(58．8％)能产生多态性。14个引物产生的112个DNA片断,用于计算种间Jaccard遗传相似性系数分析,在NTSYS程序中利用UPGMA构建系统发育树状图。分析结果表明：(1)14个Kengyilia物种存在较大的遗传多样性;(2)青藏高原的物种与新疆的物种 的RAPD变异极大;(3)形态相似、地理分布一致的物种有一定的亲缘关系,聚类在一起;(4)RAPD结果与形态学和细胞学等分析结果一致。RAPD分析方法将为Kengyilia系统分类提供DNA水平上丰富的资料。     

用RAPD技术探讨中国枣的种下划分

利用随机扩增多态性DNA(RAPD)技术对14个枣Ziziphus jujuba 品种和1个野生种——泰山酸枣Z．spinosus的遗传变异进行了研究。从120个10-碱基随机引物中筛选出37个多态性引物用于正式扩增,共扩增出429条DNA带,其中多态性带214条,占49．88％。根据DNA扩增结果计算了品种及类型间遗传距离,并用UPGMA构建了聚类树状图。分析结果表明：龙爪枣 Z．jujuba var．tortuosa、葫芦枣 Z. jujuba var．lageniformis、无核枣 Z.jujuba var．anucleatus 等几个变种内的遗传距离大于变种间遗传距离,认为枣的变种划分是不自然的,宜并入其原变种;枣种下不宜设变种,对枣种下的众多品种,应根据品种间的遗传关系,直接划分品种群。     

Phylogeny analysis of 25 apple rootstocks using RAPD markers and tactical gene tagging

RAPD (random amplified polymorphic DNA) markers were used to fingerprint eight commercially available apple rootstocks (Nertchinsk, Northern Spy, Osman, Heyer 12, M.1, M.9, M.26 and MM.106), 10 winter hardy offsprings derived from the cross of Nertchinsk x M.9, six winter hardy offsprings derived from the cross of Nertchinsk x M.26 and one winter hardy offspring derived from each of the two crosses between Osman x Heyer 12 and Northern Spy x M.1. Phylogeny analysis using parsimony allowed us to draw the genetic relationship between these lines using only RAPD markers data. The resulting cladogram was compared to the true genetic relationship between these lines in order to assess the efficiency of RAPD markers in determining accurately the phylogenetic relationship. We also developed a DNA fingerprinting system based on 13 informative RAPD loci amplified by five RAPD primers that allowed the rapid identification of apple rootstocks.     

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

Random amplified polymorphic DNA analysis and demonstration of genetic variability among bifidobacteria isolated from rats fed with raw kidney beans

A rise in bifidobacterial numbers resembling the Escherichia coli overgrowth phenomenon was observed in the rat small intestine in a feeding experiment with kidney beans. Bifidobacterial colony counts increased from 7.6 x 10(6) to 1.7 x 10(8) cfu.g-1 of intestinal tissue in the anterior part and from less than 1 x 10(5) to 2.65 x 10(8) cfu.g-1 in posterior part of the intestine. Fifteen bifidobacterial strains were purified and further analysed. Random amplified polymorphic DNA (RAPD) assays were used to genetically differentiate bifidobacterial isolates from rat gut and compare them with type strains of 20 different species from the genus Bifidobacterium. A total of 80 arbitrary decamere primers were screened with 6 isolates, and 7 primers were chosen for the final analysis. The amplified DNA bands were scored and analysed by the unweighted pair-group method using arithmetic averages clustering. The isolates were not identical to each other nor to the screened type strains. Whereas it was possible to group 12 of the isolates into 2 separate clusters, 3 strains showed no significant relatedness to any strain. The results of the RAPD analysis indicated that there was a large degree variability among the bifidobacteria in the rat gut and demonstrated the potential applicability of such an approach in the investigation of microbial diversity in complex ecosystems.     

七种蟋蟀基因组DNA的RAPD多态性研究（直翅目：蟋蟀总科）

应用10种随机引物,对西北地区常见的3属7种蟋蟀进行RAPD多态性检测,共筛选出2种引物S142,S8可以对7个种扩增出清晰稳定的多态性片段,多态性片段共计58条,相对分子质量在320bp-2400bp之间。应用UPGMA（非加权配对算术平均法）对多态性片段进行聚类分析,构建树状图,推测系统发生关系。每一种蟋蟀均先各自聚为一类,棺头蟋属与油葫芦属间的遗传距离最小,亲缘关系最近,斗蟋属4个种间的亲缘关系较为复杂,明显分为2个支系,与传统分类并不一致。     

Confirmation of relationships among Boesenbergia (Zingiberaceae) and related genera by RAPD

The relationships among 19 accessions of Zingiberaceae belonging to 11 species of Boesenbergia, six species of Kaempferia, and two species of Scaphochlamys from Southern Thailand were studied using random amplified polymorphic DNA (RAPD) profiles from leaf tissue samples. The RAPD was carried out using 10 random decamer arbitrary primers. Amplification occurred in five out of 10 tested primers (OPAM-01, OPAM-03, OPAM-12, OPB-14, OPZ-03). Total of 53 amplified bands were observed. Data obtained from the RAPD fingerprints from the samples clarified some doubts in morphological classification. The data were analyzed for the Nei and Li's Dice similarity coefficient for pair-wise comparison between individual samples and the distance matrix. The dendrogram resulting from cluster analysis, UPGMA and a principal component analysis of the RAPD result confirms a higher degree of relationship between Boesenbergia and Scaphochlamys than between Boesenbergia and Kaempferia.     

Systematic Study of Korean Formica Ants Using RAPD

ABSTRACT The phytogeny of Formica ants in Korea was carried out using RAPD. Analyzing RAPD marker, the similarity degrees between species were estimated, and they were compared with the results from the measuring values of thirteen morphological characters. Morphological analysis showed as follows: all the five species were grouped with 72% or more similarity. Out-group, Camponotus was independently isolated. F. lemani and F. Candida, F. yessensis and F.japonica were grouped in a very tight cluster with 97% or more similarity, while F. sanguinea+F. yessensis and F.japonica were grouped with 88%. On the other hand, RAPD analysis showed as followes: F. sanguinea, F. yessensis were grouped at the level of 97% similarity, F. lemani and, F. Candida were also grouped with 97%. Both two groups were again grouped with 85% or more. This group and F.japonica were grouped with 72% similarity. In RAPD the lowest similarity was 72% in the five species of Formica. The positional homology of amplified fragments was identified by Southern hybridiztion of RAPD fragments. As a result, F. sanguinea and F. yessensis showed 97% degree of similarity, and also F. lemani and F. Candida showed 97%. F.japonica and above four species showed 72%. The level of similarity of F.japonica against the other four species was lower than those of four other species.     

Mapping a cave fish genome: polygenic systems and regressive evolution

We used random amplified polymorphic DNA (RAPD) fingerprinting to generate anonymous DNA markers in the fish Astyanax mexicanus, a species with both surface and cave populations. Surface individuals are eyed and pigmented; troglobitic forms are blind and depigmented. We hybridized surface fish and Pachon population cave fish and produced a RAPD genomic map 1064 cM in length (about half the total length of the genome) that was used to screen for quantitative trait loci (QTL) for troglomorphic traits. Three QTL for reduced eye size, two for decreased numbers of melanophores, two for condition factor, and the locus for the unifactorial trait, albinism, were mapped. These factors account for an average of 46% of the variance in these traits in the backcross. The results are the first direct demonstration that troglomorphic changes in this population are multifactorial. Two closely linked pairs of QTL were found. Each consisted of a regressive and a constructive trait QTL. These close linkages are unlikely to have occurred by chance (P <.05 for each) and suggest that troglomorphic evolution might be facilitated by pleiotropy or by genetic hitchhiking.     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

用RAPD分子标记探讨沙拐枣属的种间关系

利用随机扩增多态性DNA（RAPD）技术分析了14种沙拐枣属（Calligonum L.)植物,通过对16个Sangon公司十聚体随机引物进行PCR扩增,3个引物能产生多态性带。对3个引物扩增产生的45条扩增产物,计算单匹配系数,应用UPGMA方法构建亲缘关系树状图。分析结果表明：（1）物种间遗传差异明显,具有丰富的遗传多样性;（2）14种沙拐枣属植物明显聚为4类,与传统的形态学分类结果基本一致。     

用RAPD和AFLP的方法对中国卤虫(Artemia)种及亲缘关系的研究

利用ＲＡＰＤ（随机扩增多态ＤＮＡ）和ＡＦＬＰ（扩增片段长度多态性）技术对不同种及种群卤虫的关系进行分析。 １０１个随机引物对４种卤虫Ａｆｒａｎｃｉｓｃａｎａ、Ａ ｕｒｍｉａｎａ、Ａ ｓｉｎｉｃａ和Ａ．ｐａｒｔｈｅｎｏｇｅｎｅｌｉｃａ基因组ＤＮＡ进行扩增,平均每个种获得７５１条带,其中４５８条带为多态性标记,每个引物提供平均７４个标记信息,聚类结果表明Ａ．ｓｉｎｉｃａ是不同于其他旧大陆两性生殖卤虫的一个独立的种。对来自 １５个种及品系的卤虫的 ＡＦＬＰ分析显示了非常好的遗传多态性,采用 １２对引物检测到 ５９４条带,其中 ４８０个为多态性标记。聚类结果表明来自西藏的两性生殖卤虫为不同于中国内陆两性生殖卤虫的新种。而孤雌生殖卤虫在进化过程中可能是多源的,中国内陆和沿海的孤雌生殖卤虫是沿着不同的途径进化的,内陆和沿海的孤雌生殖卤虫可能为不同的种。     

Characterization of chromosomal DNA and DNA polymorphism in Korean cultivars ofAllium sativum L.

The genetic background of the garlic (Allium sativum L.) is not well understood, since it is cultivated exclusively by vegetative propagation. To understand its genetic background, a local cultivar, Danyang, was chosen, and several basic characteristics of its chromosomal DNA were examined. Its G + C content was 40.6%, and the relative proportion of fast reassociated sequences, intermediate reassociated sequences, and slow reassociated sequences were 12%, 40%, and 48%, respectively. The genome size, calculated based on reassociation kinetic experiments, was 1.11 x 10¹⁰ bp or 12.16 pg per haploid genome. To compare the genetic variation among four local cultivars, Munkyung, Seosan, Euiseong, and Danyang, random amplified polymorphic DNA (RAPD) analysis was performed. By using slightly longer primers, 18–24 nucleotides in size, than the traditional primers used for such analysis, more reliable RAPD results were obtained. 15 primers gave rise to amplified bands, and the results could be grouped into two categories. The patterns of amplified products produced by 12 primers, group A, were polymorphic. These results were analyzed using a NTSYS-PC (Numerical Taxonomy and Multivariate Analysis System), and a dendrogram grouping the four local cultivars was produced. The three primers of group B gave rise to a monomorphic band pattern from four local garlic clutivars, indicating that these primers possibly recognize garlic specific sequences. These primers were useful in identifying genetic variations among theAllium species.     

旱麦草属种质资源的随机扩增多态性DNA（RAPDs）分析

利用３１个１０ｂｐ随机引物对来自我国新疆和中东地区的１１份旱麦草属种质资源材料以及两个普通小麦亲本和１个普通小麦与东方旱麦草的远缘杂种后代进行了ＲＡＰＤ分析。对扩增形成的３２１条谱带进行的研究发现,该属植物在新疆地区具有较丰富的遗传多样性,同时还将１个根据形态性状定名为光穗旱麦草的样品修正为东方旱麦草。研究还揭示出旱麦草属与普通小麦之间有明显的遗传分化,并发现四部体光穗旱麦草的两个基因组分别来自二     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.