江浙地区杨梅主要品种的ISSR分析

为研究江浙地区杨梅（Myrica rubra Sieb．et Zucc．）的亲缘关系,运用ISSR—PCR分析方法对其主要栽培品种进行了分析。8条引物扩增出58个DNA片段,其中多态性片段42个,占总扩增片段的72．4％。结合遗传距离分析,运用UPGMA法构建了分子系统树。结果表明,所分析的14个品种和1个优良单株以0．14为结合线可分为5组,说明江浙地区的杨梅在广泛基因交流的情况下,存在局部的隔离。‘小叶细蒂’与‘极早熟1号’亲缘关系较近,前者可能是后者的亲本之一。     

Isolation and characterization of microsatellite loci in the Amami rabbit (Pentalagus furnessi)

We report on the isolation and characterization of eight polymorphic and five monomorphic microsatellites in the Amami rabbit (Pentalagus furnessi). Microsatellite polymorphism was determined using 25 individuals. There were 2–11 alleles for each polymorphic locus with heterozygosity ranging between 0.08 and 0.76. Linkage disequilibrium was not suggested between any pairs among the eight polymorphic loci. We suggest that these primers be used in future studies to monitor population size, determine dispersal patterns, and genetic diversity within and between populations of this and related species.     

Microsatellite loci isolated from the scleractinian coral, Acropora nobilis

We report the isolation and characterization of eight microsatellite loci from the scleractinian coral, Acropora nobilis. The microsatellite loci were obtained using compound SSR primers or an enrichment protocol. All the loci were polymorphic with four to eight alleles per locus and observed heterozygosities ranging from 0.22 to 0.76. Some of the primers developed for the two congeners, Acropora palmata and Acropora millepora were applicable to A. nobilis. These loci are useful for studying the connectivity among A. nobilis populations in Okinawa, southern Japan.     

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers.

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.     

Design and implementation of degenerate microsatellite primers for the mammalian clade

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ~1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120-160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced.     

利用RAPD分析杜氏藻属（Dunaliella）嗜盐种间遗传多样性

本研究首次运用随机引物对杜氏藻属（Dunaliella）中6个嗜盐种的基因组DNA进行RAPD分析。筛选获得的6个有效引物共扩增出98个可重复的DNA片段,其中95条带具有多态性,多态性条带的频率为96．9％。根据系统进化树图将杜氏藻属6个种分别归于亲缘关系相对较远的2个类群中,且6个种与它们各自的生态分布联系不紧密。     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

水稻广亲和基因（WCG）近等基因系的随机引物PCR分析

应用随机引物ＰＣＲ（ＲａｎｄｏｍＰｒｉｍｅｒＰＣＲ）技术分别在水稻广亲和基因（ＷＣＧ）的近等基因系和色素原基因（Ｃ）的分离群体库中寻找与ＷＣＧ和Ｃ基因连锁的分子标记。对于ＷＣＧ近等基因系,在２２６个随机引物中初筛到２２个显示多态性片段的引物。根据理论值计算,在２２个多态性片段中预期有２０个与ＷＣＧ连锁。在这些连锁标记中距ＷＣＧ最近的可达０．５ｃＭ。同样在分离群体库的筛选中有１０个扩增产物与Ｃ基因连锁。     

长江、黄河流域两棉区陆地棉品种的遗传多样性比较研究

从300个随机引物中筛选到带型清晰且重复性强的41个多态性引物,用于长江、黄河流域两棉区的91陆地棉品种的RAPD标记分析。分析采用Jaccard‘s相似系数,使用NTSYS－pcl.80数据分析软件,非加权组平均法（UPGMA）聚类。来自长江流域棉区的23个陆地棉品种产生了84个多态性位点,品种之间的平均相似系数为0．631。而来自黄河流域棉区的68个陆地棉品种产生了96个多态性位点,品种之间的平均相似系数为0．632.两棉区品种的相似系数矩阵比较及成对相似系数分布的比较均表明,长江流域和黄河流域棉区陆地棉品种的遗传多样性水平相当。共同的基础种质资源、相同的育种目标及相近的育种方法和策略可能是造成两大棉区品种遗传多样性水平相当的重要因素。     

新疆吐鲁番荒漠土壤绿藻的多样性分析

应用RAPD技术对吐鲁番地区火焰山及艾丁湖区域分离的15株土壤绿藻(chlorophyta)品系的遗传多样性及其亲缘关系进行探讨。结果表明:从20个随机引物中,筛选出多态性和重复性较好且谱带清晰的引物8个,这8个引物扩增出的DNA片段大多在300～2 000 bp之间,所形成的多态性位点数差距较大,显示该区域土壤绿藻具有较丰富的遗传多样性;15株土壤绿藻扩增共得到74条谱带,71条多态性带,其多态性比率为95.95%;聚类分析显示15株土壤绿藻明显地聚为2大类,与其来源相对应,即隶属于同一亚组或相近亚组的不同种基本归为一类,其种间关系与传统的形态学分类结果相吻合。     

Isolation and characterization of microsatellite markers in red‐flanked bushrobin,Tarsiger cyanurus (Aves: Turdidae)

Microsatellite markers were isolated from red‐flanked bushrobin, Tarsiger cyanurus, for parentage analysis. From an enriched genomic library using the fast isolation by AFLP of sequences containing repeats (FIASCO) method, six polymorphic loci were obtained. Seven to 21 alleles were identified in an analysis of 19 red‐flanked bushrobin, with observed heterozygosity ranging from 0.842 to 1.00. All loci showed Hardy–Weinberg equilibrium and linkage equilibrium. Total exclusion probability using six loci was 0.998 for neither parent known, and 0.999 for one parent known. We also tested the utility of these loci on four species of Turdidae. Although one of the loci failed to amplify in any species, the other primers successfully amplified in at least two of the species we tested.     

Characterization of polymorphic microsatellite markers in the water rat (Hydromys chrysogaster)

The water rat (Hydromys chrysogaster) is well adapted to a semiaquatic life and is endemic to dispersed regions of Australia and New Guinea. To analyse the genetic diversity of water rat populations, polymorphic microsatellite markers were developed. A partial genomic library was screened for microsatellite sequences. Following isolation of the microsatellite sequences, primers were designed to amplify seven loci and of these loci, five were polymorphic. The sample tested for polymorphisms came from areas across Australia and New Guinea. Between three and 13 alleles were detected for each locus. In addition the primers amplified two loci in Mus musculus and Rattus rattus.     

Development of polymorphic nuclear microsatellite markers for polyploid and diploid members of the orchid genus Dactylorhiza

Dactylorhiza traunsteineri is an allotetraploid species that belongs to the large Dactylorhiza incarnata/maculata polyploid complex of the Eurasian genus Dactylorhiza (Orchidaceae). Here, eight polymorphic microsatellite loci were isolated using selective hybridization according to the FIASCO protocol (fast isolation by AFLP of sequences containing repeats) with slight modifications. The number of alleles per locus ranged from two to seven. All loci were possible to amplify in several other species of Dactylorhiza, using the same primers.     

Phenolic constituents and genetic profile of Hypericum perforatum L. from India

The content of hypericins (hypericin and pseudohypericin), hyperforin, and flavonoids (rutin, hyperoside, quercitrin, and quercetin) and genetic profiles of eight accessions of Hypericum perforatum L., collected from different locations in India, have been determined. The secondary metabolite content was determined using a highly selective LC/MS/MS method. Pearson and Spearman's correlation coefficient were used to investigate the relationships between the secondary metabolites and a significant positive correlation was found between hypericin and pseudohypericin contents. Genetic profiling was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) methods. Among the 49 random primers used for the initial screening, only nine yielded polymorphic RAPD profiles. The SSR analysis shows that seven out of the 11 primers were polymorphic. There exists only a partial correlation between the chemical content and genetic profiling data among the accessions under study.     

Application of RAPD markers for characterization of γ-ray-induced rose mutants and assessment of genetic diversity

Six parent and their 12 gamma ray-induced somatic flower colour mutants of garden rose were characterized to discriminate the mutants from their respective parents and understanding the genetic diversity using Random amplification of polymorphic DNA (RAPD) markers. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. The other 7 primers gave highly polymorphic banding patterns among the radiomutants. All the cultivars were identified by using only 7 primers. Moreover, individual mutants were also distinguished by unique RAPD marker bands. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. A dendrogram was constructed based on the similarity matrix using the Neighbor Joining Tree method showed three main clusters. The present RAPD analysis can be used not only for estimating genetic diversity present in gamma ray-induced mutants but also for correct identification of mutant/new varieties for their legal protection under plant variety rights.     

Eighteen microsatellite loci for endemic African weakly electric fish (Campylomormyrus,Mormyridae) and their cross species applicability among related taxa

We describe isolation and characterization of the first microsatellite loci specifically developed for African weakly electric fish (Mormyridae), for the genus Campylomormyrus. Seventeen of our 18 loci are polymorphic within the Campylomormyrus numenius species complex. The polymorphic loci showed four to 15 alleles per locus, an expected heterozygosity between 0.46 and 0.94, and an observed heterozygosity between 0.31 and 1.00. Most primers also yield reproducible results in several other mormyrid species. These loci comprise a set of molecular markers for various applications, from moderately polymorphic loci suitable for population studies to highly polymorphic loci for pedigree analysis in mormyrids.     

Rapid detection of maize DNA sequence variation.

The allele-specific polymerase chain reaction (ASPCR) has been used to determine the genotype of maize lines at two loci, wx and NPI288. The ASPCR method uses allele-specific oligonucleotide primers in PCR amplifications to amplify and discriminate simultaneously between polymorphic alleles. The success of this technique relies on the specific failure of PCR to amplify with primers that do not perfectly match the DNA sequence of one of the allelic variants. Amplification results were evaluated by dot-blot hybridization using an alkaline-phosphatase-coupled probe. The technique's speed, accuracy, sensitivity, and high throughput make it valuable for plant-breeding applications.     

光肩星天牛种群间及其近缘种遗传关系的RAPD研究

利用RAPD技术对采自中国和美国的星天牛属Anoplophora 5个种及8个光肩星天牛Anoplophora glabripennis (Motschulsky)地理种群共13个样品进行了遗传相似性分析。选用了Operon公司生产的引物H系列20个,L系列20个,Q系列11个共51个引物,最后从40个引物中筛选出26个具有多态性的引物作为第一组用于星天牛属种间和光肩星天牛种群间分析,从31个引物中筛选出19个具有多态性的引物作为第二组单独用于光肩星天牛种群分析。根据第一组引物实验获得的RAPD聚类图及遗传距离表明,各个地理种群的光肩星天牛和黄斑星天牛A. nobilis都聚在一起,形成一个大的分枝,而四川星天牛A. freyi、楝星天牛A. horsfieldi和星天牛A. chinensis均在此分枝之外。来自美国纽约和芝加哥的光肩星天牛种群聚于中国光肩星天牛种群之外的另一个独立的分枝上。分布在我国宁夏、内蒙古和河北的光肩星天牛以及宁夏黄斑星天牛和山东、陕西的光肩星天牛分别聚在一起,而甘肃的光肩星天牛与甘肃的黄斑星天牛则聚于另一枝,且它们之间的遗传距离很近,仅为0.1324,说明这两者之间有着极其相近的亲缘关系,由此推断光肩星天牛和黄斑星天牛的差异很小,遗传关系难以区分,进一步证实了它们很可能是同一个种下的两个不同的型。第二组引物实验得到了相似的结果,来自中国的6个光肩星天牛种群全部聚于同一枝中并分成两小枝： 分布于我国宁夏、河北、山东、甘肃的光肩星天牛聚在一起,内蒙古和陕西的光肩星天牛则成另一枝,而分布于美国纽约和芝加哥的光肩星天牛仍聚于中国光肩星天牛种群之外的一个单独的分枝上。但是美国光肩星天牛与中国光肩星天牛之间的遗传距离最近的为0.4578, 最远的为0.5960。由此认为,本研究中采自美国的两个光肩星天牛种群的样本和采自中国的光肩星天牛种群的样本之间存在显著差异,遗传关系较远。有必要从中国和世界其他天牛分布地采集更多样本做进一步DNA 分析。     

爪哇根结线虫Mi-毒性和无毒近等基因系的制备及其遗传变异的初步分析

爪哇根结线虫是以孤雌生殖方式繁殖的农作物重要病原物,通过将爪哇根结线虫-无毒群体在抗病番茄品种Momotaro上连续选择19代,获得了一对针对抗性基因Mi的毒性和无毒近等基因系,用102种引物对该近等基因系进行随机扩增多态性DNA（RAPD）分析显示,除一种引物外两者的RAPD带谱基本一致,证实了该毒性和无毒群体的基因组组成十分近似,对一种引物扩增出的一个无毒群体特异性的RAPD片段进行了克隆,Southern杂交结果提示该多态性片段及其同源序列在毒性选择过程中被从毒性线虫的基因组中删除,但DNA序列分析和数据库检索表明,该片段与迄今发表的核酸序列均不存在显著的同源性,因此尚无法预测其潜在的功能性,爪哇根结线虫Mi-毒性和无毒近等基因系的制备为进一步鉴定和分离该线虫毒性相关基因打下了坚实的基础。     

满天星试管苗与其玻璃化苗的RAPD指纹图谱分析

采用分离群体分组分析法(BSA),用100个随机引物对满天星的正常苗和玻璃化苗进行RAPD分析的结果表明,7个随机引物扩增出多态性差异条带。再用上述7个引物分别对试管苗及其玻璃化苗个体进行DNA的PCR扩增的结果显示,引物J20在2种苗中出现差异条带。