应用RAMP标记分析国产姜黄属植物的遗传多样性

本研究运用RAMP(random amplified microsatellite polymorphism)分子标记分析了10种国产姜黄属(Curcuma)植物的遗传变异和亲缘关系。结果得到520条迁移率不同的条带,其中多态性条带有509条,每条引物平均得到10.6条带和10.4条多态性带,多态性条带的百分率为97.88%。69份供试材料之间遗传相似系数最大值为0.921,最小值为0.550。当遗传距离是0.61时,可将其分为四大类群。研究结果表明种间关系与地理位置有一定的关系,同时证实了姜黄属植物种质资源遗传多样性丰富,为姜黄属植物的分类鉴定奠定了基础。     

观叶福禄桐遗传多样性的ISSR分析

利用ISSR分子标记对9个观叶福禄桐品种进行遗传多样性和亲缘关系分析,从100条引物中筛选出9条稳定、多态性高的引物用于PCR扩增,共获得70条带,其中多态性条带61条,多态百分率为87.14%。聚类结果显示,品种间相似性系数为0.346 9-0.816 3,聚类结果与品种间的地理来源紧密相关,从外观形态上比较,亲缘性较近的叶形和株型相似性较高;观叶福禄桐各品种间基因型差异较小,亲缘关系较近,遗传基础相对较窄。     

鸢尾属部分植物种质资源的RAPD分析

采用RAPD分子标记技术,从100个随机引物中筛选出多态性强、重复性好且稳定性高的引物18个,对38份野生鸢尾属材料进行扩增,共扩增出409条带,其中多态性带405条,多态性比率为99.0%,表明野生鸢尾属植物种间有丰富的遗传多态性;根据DNA谱带计算物种间遗传距离,聚类分析结果将鸢尾属38份材料划分为6组,其结果与传统生物学特性划分的6个亚属的分类结果基本一致;物种特有RAPD标记分析表明,利用18个引物可以较好地将鸢尾属38种植物区分开,其中9个材料得到了单一标记的扩增带,表明运用RAPD分子标记对研究鸢尾属植物特异性基因及标记的筛选等有一定的理论和实际应用价值。     

应用ISSR标记研究仲彬草属植物的遗传变异

利用ISSR标记对仲彬草属14个种和1个变种共32份材料进行了研究。结果表明:仲彬草属材料间ISSR标记多态性较高,变异较大。12个引物共扩增出593条带,其中535条(90.2%)具有多态性,每个引物可扩增出11~80条多态性带,平均44.6条。ISSR标记遗传相似系数在种间变化范围为0.430~0.866,平均值为0.620。从聚类分析得知,ISSR标记能将32份仲彬草属材料完全分开,32份材料聚为4类。同种不同居群的材料分别聚在一起,亲缘关系较近,同时,在分子水平上种内不同居群间也存在分化;种间存在明显的遗传差异;形态相似、地理分布一致的物种倾向于聚类在一起,有一定的亲缘关系。因此,ISSR分子标记能有效地评价仲彬草属物种的亲缘关系。     

国产甘草属植物的RAPD分析及其分类学研究

应用RAPD技术,探讨甘草属(G ly cy rrh iza L.)13种1变种30个植物类群的遗传差异和几个争议种的分类地位。从60个随机引物中筛选出14个多态性好的引物进行RAPD实验,DNA片段的二态数据用U PGM A聚类法构建系统发育树。共扩增出250条带,多态性带204条,约占总数的81.7%。聚类结果显示RAPD分子标记构建的系统发育树与经典分类系统一致。甘草属植物具有丰富的遗传多样性,同种内不同居群间的遗传分化较大。黄甘草、胀果甘草、乌拉尔甘草三者亲缘关系较近,平卧甘草与粗毛甘草存在很大的遗传差异,作为独立种较合理。RAPD标记可为甘草属植物的系统分类研究提供分子生物学依据。     

松花型花椰菜主要品种鉴定的分子标记分析

利用RAPD、ISSR和SRAP 3种分子标记对我国南方地区松花型花椰菜主栽品种进行鉴定,分析了品种间的遗传多样性。3种标记共产生370条扩增带,238条为多态性条带,其多态率为64.32%。其中只有SRAP标记的引物m e1/em1可将20个品种全部鉴别。遗传相似系数分析表明,松花型花椰菜品种之间的亲缘关系较近,遗传背景比较狭窄。聚类分析表明品种间的亲缘关系与熟性、地理分布相关。研究表明,分子标记能有效地应用于花椰菜品种鉴定,且综合多种分子标记分析品种间的遗传多样性将更加准确可靠。     

10份续断属植物亲缘关系的ISSR分析（英文）

续断属(Dipsacus L.)植物是中国续断科(Dipsacaceae)传统中药材,但属下种间亲缘关系仍存在不准确性鉴定。本研究拟采用10条多态性较高的ISSR(Inter-simple sequence repeat)标记从分子水平研究10份续断属植物的分类和种间亲缘关系,为该属植物的科学分类提供资料。从100条ISSR引物中筛选出多态性较高的10条,对10份续断材料进行PCR扩增,共扩增出947条DNA带,其中多态性条带828条,平均多态性条带百分率为87.4%,表明续断属植物具有丰富的遗传变异。遗传相似系数变化范围在0.557~0.806,平均为0.661。聚类分析结果表明,10份续断在相似系数水平0.582处可分为2个大类,进一步在0.702又可分为5个小类群。第1大类群包括3个小类群,第1小类群包括4分大理续断[DL(L),DL(W),DL(H),DL(WS)],第2小类群包括日本续断(RB)和恩施续断(ES),第3小类群包括2份川续断[C(J)和C(D)],这表明小类群间品系亲缘关系比较近,基因交流比较频繁;第2大类群包括大头续断(DT)和深紫续断(SZ),两者遗传距离较远,分别构成另外两个小类群。主成份分析与聚类分析所得的结果基本一致。研究结果还发现日本续断(RB)和恩施续断(ES)两者亲缘关系较近(0.210 1),且两者的地理分布有较大的重叠,可能两者基因交流比较频繁,由于环境的不同形态上稍有差异。推测恩施续断可能为日本续断的一个变种,当然这还需要进一步深入研究。     

中国红树科7种红树植物遗传多样性分析

以红树、红海榄、秋茄、角果木、木榄、海莲、尖瓣海莲等7种红树科植物为材料,采用改进的CTAB法获得了纯度较高、得率高、片段完整的基因组DNA。通过筛选出的15个有效引物进行RAPD分析,探讨了7种树植物间的亲缘关系。15个有效引物共扩增出617条DNA带,其中多态性条带415条,占总扩增条带的67.26%。利用Nei指数法得出7个分类群间的遗传一致度和遗传距离,并运用UPGMA法进行聚类分析。7个分类群分为A、B两个大组,平均遗传距离为0.41。将得出的7个分类群的DNA分子分类系统图,与传统的分类进行比较,发现结果相符。同时获得一个OPG05－900的差异片段可作为区分海莲和尖瓣海莲的分子标记。     

51个春兰(Cymbidium goeringii)品种的AFLP遗传多样性分析

为了揭示春兰品种的遗传多样性和亲缘关系,为春兰种质资源的有效利用和开发提供依据,采用AFLP技术对51个春兰品种进行了遗传多样性分析,经筛选得到了8对条带清晰、多态性高的引物,共扩增出1315条DNA片段,其中多态性条带为1217条,平均1对引物扩增条带164条,多态性带152条,多态性位点频率为92.5%,表明春兰品种具有丰富的遗传多态性。49个品种含有特有带。51个品种间遗传相似系数变化范围为0.501~0.716,聚类分析表明,51个春兰品种共分为5个类群,来自同一地区的品种并没有聚在一起,表明春兰品种的遗传背景混乱。AFLP分子标记技术能有效地分析春兰品种的遗传多样性和亲缘关系。     

七种风铃草属植物亲缘关系的ISSR分析

为了解四川风铃草属植物的资源丰富度和遗传进化情况,该研究利用ISSR分子标记,探究了四川7种风铃草属植物的亲缘关系,为风铃草属植物的分子标记辅助鉴定、资源保护、花卉品种的开发与育种提供理论基础。结果表明:40条ISSR引物中有28条引物能够扩增出清晰的条带,扩增总条带数为164,其中有98.8%的扩增条带具有多态性,供试的7种风铃草属植物遗传相似性系数在0.421～0.945之间,其中钻裂风铃草与藏滇风铃草的遗传相似度最高为0.945,说明它们之间的亲缘关系很近。此外,ISSR分子标记聚类结果表明,7种风铃草属植物可以明显聚为4大类:西南风铃草、灰毛风铃草、灰岩风铃草为一类;紫斑风铃草与流石风铃草各成一类,这两种在形态上与其它各种风铃草差异较大;最后是钻裂风铃草与藏滇风铃草为一类。     

Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats

The abundance and scattered distribution of simple-sequence repeats (SSR) in eukaryotic genomes prompted us to explore the use of SSR-based oligonucleotide primers in single primer amplification reactions. In a pilot experiment, 23 primers were used across a panel of evolutionarily diverse eukaryotic genomes, including grapes, lettuce, tomato, pine, maize, salmon, chicken, Holstein cows and humans. The primers were 16–20 bases in length and represented SSRs of di-, tri-, tetra-, and pentanucleotide repeats. The results showed that tetranucleotide repeat primers were most effective in amplifying polymorphic patterns. Of 11 such primers tested, 70% produced polymorphic patterns from the DNA of one or more species. Primers representing a combination of two tetranucleotide repeats, or compound microsatellites, were equally effective. The polymorphisms contained in such fingerprints were able to identify individuals of vertebrate species as well as lines or varieties of plants. Inheritance of the polymorphic bands was studied in a maize recombinant inbred population, DE811 x B73. Thirty-two polymorphic bands, derived from two amplification patterns, were mapped as dominant markers on an existing RFLP map of the same population. The bands were distributed across nine of the ten chromosomes.     

Restriction Endonuclease Analysis of Highly Repetitive DNA as a Phylogenetic Tool

Multiple band patterns of DNA repeats in the 20–500-nucleotide range can be detected by digesting genomic DNA with short—cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA ``taxonprints' (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects—altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies. Received: 10 February 1997 / Accepted: 10 March 1997     

Separation and purification of small quantitites of specific RNA species by polyacrylamide gel electrophoresis using prestained RNAs as markers.

A method is described for the separation and purification of different molecular species of RNA in microquantities using polyacrylamide gel electrophoresis. The experimental procedure consists of the following steps; (i) partial prestaining of RNA with methylene blue at a concentration of the dye which would not affect the electrophoretic migration of the RNA bands, but which would permit visual observation of the migrating bands during electrophoresis; (ii) use of short columns just sufficient to achieve separation of each species of RNA as a single compact band rather than a series of bands; (iii) trapping of each of the eluting RNA species from the gel on DEAE-cellulose dises in sequential order; and (iv) elution of the RNAs from the DEAE-discs by extraction with triethylammonium bicarbonate and recovery of the corresponding RNAs by lyophylization.     

Evaluation of copper-induced stress on eggplant (Solanum melongena L.) seedlings at the molecular and population levels by use of various biomarkers

Heavy-metal contamination is an important environmental problem in the world. It is known that high concentrations of heavy metals cause toxic damage to cells and tissues. In this study the effects of copper (Cu(2+)) contamination were determined at the molecular and population levels in eggplant (Solanum melongena L.) seedlings exposed to various concentrations of the metal ion. Inhibition of root growth, reduction in dry weight and total soluble protein content in the roots of eggplant seedlings were observed with increasing Cu(2+) concentrations. In ecotoxicology, analysis by random amplification of polymorphic DNA (RAPD) has been applied as a suitable biomarker assay for plants. For the RAPD analyses, nine RAPD primers were found to produce unique polymorphic band patterns and were subsequently used to produce a total of 80 and 168 bands in the roots of untreated and treated eggplant seedlings, respectively. The changes in RAPD profiles after Cu(2+) contamination were considered as variations, i.e. as gain and/or loss of bands compared with control seedlings. These results suggest that changes in genomic template stability could be detected with RAPD profiles and this result could be compared with the growth, dry weight and total soluble protein content of the seedlings grown at various Cu(2+) concentrations. The measurements of parameters at the molecular and population levels are fundamental to accumulate valuable information and to understand clearly the effect of a contaminant on an organism in ecotoxicology.     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach

A new chemotaxonomic method is presented for the identification of eubacteria. This method is based on one-dimensinal gel electrophoresis of total RNA extracts from eubacteria. Only low molecular weight (<150 nucleotides) RNA, comprising 5S ribosomal and transfer RNA, was used for the identification. The high resolution of the electrophoresis, better than half a nucleotide, allowed construction of low molecular weight (LMW) RNA profiles that contained 10 to 20 bands per strain. LMW RNA profiles of a set of eubacterial reference strains showed on variation in dependence on culture conditions or physiological state of the cells. Computer-assisted data evaluation, including six molecular weight markers, enabled the calculation of relative nucleotide units (RNU) for every band. The resulting normalized band pattern allowed the identification of identical strains on different gels. The relative position of the single bands from the different groups of RNAs made an identification of bacterial strains to genus and often species level possible.Especially valuable for the identification were the large, class 2 tRNAs that showed certain variation among species of the same genus and varied considerably among different genera. RNA profiles can provide a rapid and inexpensive screening technique for the taxonomic classification of single bacterial strains. Potential fields of application for this technique might be bacterial taxonomy, biotechnology and ecology.     

A feasibility study of the use of random amplified polymorphic DNA in the population genetics and systematics of grasshoppers.

Single, short primers of arbitrary nucleotide sequence were used in polymerase chain reactions to amplify regions of DNA isolated from several melanopline and oedipodine grasshoppers collected from local Saskatchewan populations. This represents one of the first applications of the method, called randomly amplified polymorphic DNA (or RAPD), to natural populations. Twenty-four different oligonucleotide primers, nine nucleotides in length, yielded clear and reproducible bands corresponding to amplified products and separable by agarose gel electrophoresis. On average, about 8.1 bands (range 0-17) were obtained per primer per individual. The mean percent similarity between band profiles of conspecific individuals was 51.2%, whereas the mean value for individuals representing different species or genera was 35.0%. Clearly, greater numbers of insects and primers will be required to achieve a satisfactory level of phylogenetic resolution. Given RAPDs technical advantages and ease of execution, however, this should not be problematic to the molecular systematist.     

Sequence homology of polymorphic AFLP markers in garlic (Allium sativum L.).

Linkage mapping and genetic diversity studies with DNA markers in plant species assume that comigrating bands are identical, or at least that they have homologous sequences. To test this assumption in a plant with a large genome, sequence identities of 7 polymorphic amplified fragment length polymorphism (AFLP) markers of garlic, previously used to estimate similarity in genetic diversity studies, were characterized. Among 37 diverse garlic clones, 87 bands from these 7 polymorphisms were excised, amplicons were cloned, and 2 to 6 colonies were sequenced from each band, to yield a total of 191 DNA amplicons. Of these 87 bands, 83 bands (95.4%) contained AFLP amplicons that were identical or highly homologous to the typical marker of that band; only 4 bands contained amplicons with little homology to the same-sized amplicons of other garlic clones. Of these 83 bands, 64 (73.6%) contained only highly homologous amplicons (>90% sequence identity), whereas 19 (21.8%) contained both homologous and nonhomologous amplicons, with sequence identities less than 60%. Of the 37 nonhomologous amplicons identified, 25 (67.5%) differed in length from other amplicons in the band. Sequence conservation of AFLP amplicons followed patterns similar to phylogenetic relationships among garlic clones, making them useful for developing simple PCR-based markers in genetic mapping and diversity assessment.     

Genomic relations among two non-mangrove and nine mangrove species of Indian Rhizophoraceae

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to study the genomic relationship among 11 members of Indian Rhizophoraceae represented by nine true mangroves and two non-mangrove species. The AFLP and RAPD bands were scored and analyzed for genetic similarities and cluster analysis was done which separated the 11 species studied into two main groups, the true mangroves and the non-mangroves. The polymorphism observed for these markers showed a high degree of genetic diversity among the constituent taxa of the family. The phylogenetic relationship inferred from molecular marker systems supported the traditional taxonomic classification of the family Rhizophoraceae based on morphological characters at the levels of tribe, phylogeny and delimitation of genera and species, except the intra-generic classification of the genus Bruguiera and the placement of Rhizophora in the family Rhizophoraceae.