TRAP及SSCP检测草鱼微卫星序列多态性

目的：在草鱼不同种群遗传结构比较中,应用一些来自鲤鱼的微卫星引物扩增草鱼的相关序列缺少多态性,该研究通过应用几种方法提高这些序列在遗传学研究中的价值。方法：运用TRAP随机引物与鲤鱼的微卫星引物组合,扩增出多态性好、重复性强的产物;同时采用SSCP技术对那些没有长度多态的微卫星序列进一步分析,寻找这些序列存在一些核苷酸位点的多态性。结果：TRAP可以检查到多态性好、重复性强的产物;SSCP发现一部分没有长度多态性的微卫星序列存在核苷酸位点多态性并且可以清楚分辨不同的基因型。结论：结果表明TRAP及SSCP在种群遗传学研究有良好的应用前景。     

High throughput isolation of microsatellites in hop (Humulus lupulus L.)

Procedures to generate usable microsatellite marker sequences should be optimized for cost-effectiveness in each species. For hop, we have used a cocktail of several restriction enzymes to digest the genomic DNA. This is followed by capture of microsatellite-containing sequences with long microsatellite probes attached to a membrane. The enrichment level for GA and GT libraries was 37% and 35%, respectively, and 100% of the clones contained microsatellite sequences. Libraries can be generated from genomic DNA in approximately 10 d.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

Fosmid library construction and end sequences analysis of the Pacific oyster,Crassostrea gigas

The Pacific oyster (Crassostrea gigas) is globally distributed and is one of the most commercially and ecologically important marine organisms. However, little is known about the genome of this species. In this study, a C. gigas fosmid library was constructed that contains 459,936 clones with an average insert size of approximately 40 kb, representing 22.34-fold haploid genome equivalents. End sequencing generated 90,240 fosmid end sequences (FESs) with an average length of 384.27 base pairs (bp), covering approximately 2.58% of the Pacific oyster genome. The FESs were subsequently assembled and annotated, resulting in 6332 sequences with predicted open reading frames≥300 and 1,189,100 bp repeats. Furthermore, a total of 3200 microsatellite repeats were identified, and dinucleotide repeats were found to occur most abundantly, with AG and AAT being the most abundant repeat class of dinucleotides and trinucleotides. We also found that the repeat number was generally negatively proportional to the repeat element length. Microsatellites composition between the transcribed sequences and genomic sequences was shown to be different. Point mutations of microsatellite were non-random and underwent strong selection stress. Overall, a comprehensive sequence resource for the Pacific oyster was created, including annotated transposable elements, tandem repeats, protein coding sequences and microsatellites. These initial findings will serve as resources for further in-depth studies of physical mapping, gene discovery, microsatellite marker developing and evolution studies.     

Polymorphic microsatellite loci of the rat (Rattus norvegicus)

The EMBL and GenBank DNA databases were searched for microsatellite sequences of the rat containing dinucleotide repeats of (CA)_n and (GA)_n. Among those obtained, 23 sequences were analyzed by polymerase chain reaction to examine the size variation of the amplified fragment in inbred rat strains. All of the 23 microsatellite sequences varied in size among the strains tested. The 23 microsatellite loci in a pair of substrains separated from the same progenitor strain were then analyzed. Fragments identical in size were observed in all loci of the two substrains, indicating the stability of the microsatellite over a large number of generations. The microsatellite loci, therefore, should be useful markers for linkage analyses in the rat.     

商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响

两栖类有尾目物种的微卫星分离中的筛选成功率常常较低。为探索微卫星结构对筛选效率的影响,本研究通过AFLP快速分离法(fast isolation by AFLP of sequences containing repeats,FIASCO)对商城肥鲵(Pachyhynobius shangchengensis)二碱基重复类型和四碱基重复类型微卫星进行分离,并对微卫星序列进行了分析。研究中发现二碱基微卫星位点多以微卫星DNA家族形式存在,并因此导致了微卫星位点分离较低的筛选率;在四碱基重复的微卫星位点中未发现微卫星DNA家族的存在。对研究中得到的3个微卫星DNA家族的分析发现,同一家族的上、下游侧翼序列变异程度存在差异;毗邻微卫星重复单元区的侧翼序列碱基变异程度较高,而较远处的区段则相对保守。这些结构特征可能反映出微卫星DNA家族在演化中的复杂性。本文的研究结果提示在两栖动物的一些类群中,微卫星的筛选必须考虑微卫星DNA家族的影响,选取适宜的碱基重复类型将是决定筛选效率的关键。     

MICAS: a fully automated web server for microsatellite extraction and analysis from prokaryote and viral genomic sequences

MICAS is a web server for extracting microsatellite information from completely sequenced prokaryote and viral genomes, or user-submitted sequences. This server provides an integrated platform for MICdb (database of prokaryote and viral microsatellites), W-SSRF (simple sequence repeat finding program) and Autoprimer (primer design software). MICAS, through dynamic HTML page generation, helps in the systematic extraction of microsatellite information from selected genomes hosted on MICdb or from user-submitted sequences. Further, it assists in the design of primers with the help of Autoprimer, for sequences containing selected microsatellite tracts.     

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.     

中国龙虾微卫星标记的筛选及遗传多样性分析

文章以M13通用引物和重复序列(CT)₁₅、(AT)₁₅引物, 利用PCR法对中国龙虾(Panulirus stimpsoni Hoehuis)部分基因组DNA文库进行筛选。共获得78个微卫星序列, 分别分布于55个阳性重组克隆中, 其中完美型(perfect)共50个, 占64%; 非完美型(imperfect)3个, 占3.8%; 混合完美型(compound perfect)6个, 占7.7%; 混合非完美型(compound imperfect)19个, 占24.5%。根据微卫星序列, 设计并筛选出15对微卫星多态性引物, 对中国龙虾的群体进行了遗传多样性分析。获得3~12个等位基因, 等位基因大小在78~425 bp之间, 基本符合引物设计的理论长度。期望杂合度范围为0.48~0.87, 平均值为0.71, 表明中国龙虾基因组微卫星具有较高的杂合度与遗传多样性。15个微卫星位点的PIC值从0.44到0.84, 平均值为0.60, 说明这些微卫星位点在中国龙虾基因组中包含丰富的遗传信息, 合适用于中国龙虾的各种分子标记及遗传学分析和应用。     

Molecular identification of hybrids of the invasive gibel carp Carassius auratus gibelio and crucian carp Carassius carassius in Swedish waters

Both mitochondrial DNA sequence and two nuclear microsatellite markers were used to confirm the identity of the first record of Carassius auratus gibelio in the western (Swedish) Baltic Sea region. A total of 49 fishes were analysed, where 22 were from three Swedish sites connected to the Baltic Sea. The D-loop mitochondrial DNA sequences showed that 16 of 22 Swedish fishes were related to C. a. gibelio. The phylogenetic analysis of these sequences showed that these fish are probably not native, but represent different lineages of C. a. gibelio from China, Japan and Russia. All except three of these 16 fishes had microsatellite alleles suggesting hybridization with Carassius carassius. These findings suggest that a cryptic invasion of C. a. gibelio might be in progress.     

Exploiting dinucleotide microsatellites conserved among mammalian species

Dinucleotide microsatellites are useful for gene mapping projects. Depending upon definition of conservation, published estimates of dinucleotide microsatellite conservation levels vary dramatically (30% to 100%). This study focused on well-characterized genes that contain microsatellites in the human genome. The objective was to examine the feasibility of developing microsatellite markers within genes on the basis of the assumption of microsatellite conservation across distantly related species. Eight genes (Gamma-actin, carcinoembryonic antigen, apolipoprotein A-II, cardiac beta myosin heavy chain, laminin B2 chain, MHC class I CD8 alpha chain, c-reactive protein, and retinoblastoma susceptibility protein) containing large dinucleotide repeat units (N ≥ 15), complete genomic structure information, and homologous gene sequences in a second species were selected. Heterologous primers were designed from conserved exon sequences flanking a microsatellite motif. PCR products from bovine and porcine genomic DNA were tested for the presence of microsatellite sequences by Southern blot hybridization with biotin-labeled (CA)₁₂ oligonucleotides. Fragments containing microsatellites were cloned and sequenced. Homology was verified by sequence comparisons between human and corresponding bovine or porcine fragments. Four of sixteen (25%) cross-amplified PCR products contained dinucleotide repetitive sequences with repeat unit lengths of 5 to 23. Two dinucleotide repetitive sequences showed microsatellite length polymorphism, and an additional sequence displayed single-strand conformational polymorphism. Results from this study suggest that exploitation of conserved microsatellite sequences is a useful approach for developing specific genetic markers for comparative mapping purposes. Received: 7 July 1995 / Accepted: 28 September 1995     

Evolutionary Dynamics of Microsatellite Distribution in Plants: Insight from the Comparison of Sequenced Brassica,Arabidopsis and Other Angiosperm Species

Despite their ubiquity and functional importance, microsatellites have been largely ignored in comparative genomics, mostly due to the lack of genomic information. In the current study, microsatellite distribution was characterized and compared in the whole genomes and both the coding and non-coding DNA sequences of the sequenced Brassica, Arabidopsis and other angiosperm species to investigate their evolutionary dynamics in plants. The variation in the microsatellite frequencies of these angiosperm species was much smaller than those for their microsatellite numbers and genome sizes, suggesting that microsatellite frequency may be relatively stable in plants. The microsatellite frequencies of these angiosperm species were significantly negatively correlated with both their genome sizes and transposable elements contents. The pattern of microsatellite distribution may differ according to the different genomic regions (such as coding and non-coding sequences). The observed differences in many important microsatellite characteristics (especially the distribution with respect to motif length, type and repeat number) of these angiosperm species were generally accordant with their phylogenetic distance, which suggested that the evolutionary dynamics of microsatellite distribution may be generally consistent with plant divergence/evolution. Importantly, by comparing these microsatellite characteristics (especially the distribution with respect to motif type) the angiosperm species (aside from a few species) all clustered into two obviously different groups that were largely represented by monocots and dicots, suggesting a complex and generally dichotomous evolutionary pattern of microsatellite distribution in angiosperms. Polyploidy may lead to a slight increase in microsatellite frequency in the coding sequences and a significant decrease in microsatellite frequency in the whole genome/non-coding sequences, but have little effect on the microsatellite distribution with respect to motif length, type and repeat number. Interestingly, several microsatellite characteristics seemed to be constant in plant evolution, which can be well explained by the general biological rules.     

Identification of 10 882 porcine microsatellite sequences and virtual mapping of 4528 of these sequences

A total of 10 882 porcine microsatellite repeats were identified in genomic shotgun sequences from the Sino-Danish Pig Genome Sequencing Consortium (http://www.piggenome.dk). Of these, 4528 microsatellites were placed on a pig-human comparative map by blast analysis of porcine sequences against the human genome (blast cut-off threshold =1 x 10(-5)). All microsatellite sequences placed on the comparative map are accessible at http://www.animalgenome.org/QTLdb/pig.html. These sequences increase the number of identified microsatellites in the porcine genome by several orders of magnitude. They are a new resource of microsatellite sequences for generating markers to be used in linkage studies and in fine mapping and positional cloning of quantitative trait loci.     

Polymorphic microsatellite loci for the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and some remarks on their isolation

Five polymorphic tri‐ and tetranucleotide microsatellite loci suitable for population genetic analysis were identified in the cotton bollworm Helicoverpa armigera from two partial phagemid genomic libraries enriched for microsatellite inserts. The overall microsatellite‐cloning efficiency in H. armigera is 2.5%, which is approximately eightfold lower than that for the gadoid fishes (20%) employing the same enrichment protocol, supporting the notion of a relative low frequency of microsatellite sequences in lepidopteran genomes. In addition, a large proportion of cloned microsatellite sequences turned out to be repetitive DNA, thus further increasing the difficulty of developing such markers in butterflies and moths.     

Putative in silico mapping of DNA sequences to livestock genome maps using SSLP flanking sequences

In this study, an in silico approach was developed to identify homologies existing between livestock microsatellite flanking sequences and GenBank nucleotide sequences. Initially, 1955 bovine, 1570 porcine and 1121 chicken microsatellites were downloaded and the flanking sequences were compared with the nr and dbEST databases of GenBank. A total of 74 bovine, 44 porcine and 37 chicken microsatellite flanking sequences passed our criteria and had at least one significant match to human genomic sequence, genes/expressed sequence tags (ESTs) or both. GenBank annotation and BLAT searches of the UCSC human genome assembly revealed that 38 bovine, 13 porcine and 17 chicken microsatellite flanking sequences were highly similar to known human genes. Map locations were available for 67 bovine, 44 porcine and 21 chicken microsatellite flanking sequences, providing useful links in the comparative maps of humans and livestock. In support of our approach, 112 alignments with both microsatellite and match mapping information were located in the expected chromosomal regions based on previously reported syntenic relationships. The development of this in silico mapping approach has significantly increased the number of genes and EST sequences anchored to the bovine, porcine and chicken genome maps and the number of links in various human-livestock comparative maps.     

Comparative and physical mapping of 111 previously reported and 105 new porcine microsatellites

Here we report radiation hybrid mapping of 105 new porcine microsatellite markers on the IMpRH₇₀₀₀ radiation hybrid panel. In addition, we searched flanking sequences of these markers, as well as 673 previously reported RH-mapped microsatellite markers, for orthology to human sequences. Eighty-seven new and 111 previously mapped sequences exhibited orthology to human sequences. Using a stringent sequence alignment, 25 microsatellite-flanking sequences were found to be highly similar to genic sequences, whereas 173 were similar to non-genic sequences in the human genome. Five markers were located near known breakpoints of synteny between human and swine.     

巴氏蘑菇微卫星序列的分离及其对Co60辐射诱变菌株的鉴别

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。     

文蛤微卫星DNA的筛选及其特性分析

采用磁珠富集分离法从文蛤(Meretrix meretrix)的基因组中筛选得到49条微卫星DNA序列,其中两碱基重复有3种类型36个序列,四碱基重复有14种类型26个序列.重复次数在5～30之间的序列占75.6%,30次以上重复的序列占24.4%,最高重复次数为100.根据重复单元的排列特点,完美型、非完美型及混合型序列所占比例分别为61.2%、14.5%和24.5%.本研究中构建的文蛤微卫星文库将在文蛤种质资源评价及分子遗传学研究中发挥作用.     

Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans

Abstract We describe the identification of polymorphic microsatellite loci in the pathogenic yeast, Candida albicans . A search for all coding-region microsatellites with more than four repeats that can be found in Candida sequences in GenBank was conducted. Nine such microsatellite sequences consisting of trinucleotide motifs were found. Three of these were perfect microsatellites while the remaining six sequences were found in one imperfect microsatellite and two compound microsatellites. Because of the close proximity of some of these repeats, all could be assayed with six PCR primer pairs. All of these microsatellite sequences were found in five nuclear genes, ZNF1, CCN1, CPH1, EFG1 , and MNT2 . Except for a single (CTT)₅ serine tract, all coded for polyglutamine tracts. Another locus with seven alleles, a region of the ERK1 protein kinase gene, was also examined, and may be a representative of a new class of highly polymorphic ‘clustered’ microsatellites. Such loci, in which several non-contiguous but closely linked microsatellites are clustered together, may be a useful source of DNA polymorphisms in microorganisms in which long microsatellite sequences are unavailable. All seven regions amplified were polymorphic, having between two and seven variable length alleles in the 11 strains of Candida albicans examined. The results of this and similar searches will facilitate epidemiological and evolutionary studies of Candida and other microorganisms.     

Development and diversity of microsatellite markers for endangered species,Morus boninensis Koidz., to establish conservation program

We have developed seven microsatellite markers from an enrichment library of genomic DNA for an endangered species, Morus boninensis. A total of 112 of the 320 clones were found to have unique sequences with microsatellite repeats. Seven of 54 primer pairs revealed clear chromatograms and polymorphisms among 36 individuals sampled from three of the Bonin Islands. Seven to 17 alleles per locus were detected, and the expected heterozygosity without considering double reduction ranged from 0.429 to 0.819. These findings should be useful for those studying the conservation genetics of M. boninensis.