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。     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers for the guava rust fungus, Puccinia psidii

We developed and characterized 15 polymorphic microsatellite markers present in the genome of the guava rust fungus, Puccinia psidii. The primers for these microsatellite markers were designed by sequencing clones from a genomic DNA library enriched for a simple sequence repeat (SSR) motif of (AG). All these 15 primer pairs successfully amplified DNA fragments from a sample of 22 P. psidii isolates, revealing a total of 71 alleles. The observed heterozygosity at the 15 loci ranged from 0.05 to 1.00. The SSR markers developed would be useful for population genetics study of the rust fungus.     

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。     

磁珠富集法开发北柴胡多态性简单序列重复标记

目的:利用磁珠富集法分离北柴胡微卫星序列,以开发北柴胡微卫星引物,获得有多态性的简单序列重复(SSR)标记。方法:用生物素标记的混合探针(AC)15、(AG)15、(MAB)12和两端连接已知序列人工接头的北柴胡基因组DNA酶切片段混和后与磁珠杂交,构建微卫星序列富集的小片段插入文库;利用接头引物分别与生物素探针引物Biotin-(AC)15、Biotin-(AG)15、Biontin-(MAB)12形成3个组合,用PCR方法对文库进行初步筛选;对可能的阳性克隆子进行测序复筛,选取微卫星侧翼序列足够长的序列设计引物,用荧光标记的基因分型技术以栽培柴胡种质为材料分析其多态性。结果:开发了5对多态性SSR标记,它们在5份柴胡栽培种质中共扩增出30.70个多态性等位基因,平均每条引物可以扩增出6.14个多态性等位基因;观察等位基因数最多13个,最少3个;有效等位基因数最多11.4个,最少1.6个。同时分析了4对EST-SSR引物,比较了2种SSR标记扩增结果。结论:磁珠富集法是开发柴胡多态性SSR标记的有效方法。     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ~25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

随机扩增杂交微卫星方法在大仓鼠种群遗传多态性研究中的应用

随机扩增杂交微卫星(Random amplified hybridization microsatellites,RAHM)是一种复合RAPD扩增和寡核苷酸扫描的方法。该方法能够从RAPD产物凝胶上获得更多的信息,具有分析方法快速,高敏感性,能检测到高水平的多态性等优点。RAHM方法通过对RAPD扩增的DNA片段进行微卫星杂交来替代限制性内切酶对基因组DNA的消化,有助于揭示微卫星基因组克隆,进行微卫星引物的筛选。本文采用随机扩增杂交微卫星方法来检测小型哺乳动物大仓鼠(Techerskia triton)种群的遗传多态性,结果表明RAHM方法能够检测到大仓鼠种群中较高的多态性以及种群间的差异,这些条带模式可能代表真核基因组中另一种多态性标记的来源,可用于检测小型哺乳动物种群的遗传多态性。     

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an approximately 25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.     

Characterization of novel microsatellites from Drosophila transversa

We investigated a partial genomic library of Drosophila transversa for microsatellites and developed 12 markers for genetic analyses. This is the first time that microsatellite primers from the quinaria species group have been described. Four loci were cross-amplified in D. phalerata. Nine out of the 12 microsatellite markers developed are likely to be on the X chromosome.     

Microsatellite markers from a microdissected swine chromosome 6 genomic library

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting

Many molecular marker techniques are available today. PCR-based approaches are in demand because of their simplicity and requirement for only small quantities of sample DNA. Nonanchored inter simple sequence repeats (ISSRs) are arbitrary multiloci markers produced by PCR amplification with a microsatellite primer. They are advantageous because no prior genomic information is required for their use. We found the technique stable across a wide range of PCR parameters. Polymorphisms were abundant among 7 dicot species tested with 2 tri-nucleotide and 2 tetra-nucleotide primers. Thus, nonanchored ISSR markers are a good choice for DNA fingerprinting.     

Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs)

Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%–88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.     

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA · GT)>12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of >100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers U25689 and U25690.     

Isolation and characterization of microsatellite loci in the black rat snake (Elaphe obsoleta)

We obtained molecular markers useful for population level studies of the black rat snake (Elaphe obsoleta) by screening genomic DNA libraries enriched for dinucleotide, tetranucleotide, and pentanucleotide microsatellite repeats. Following sequencing of the positive clones, 11 pairs of primers were designed for polymorphic loci and their variability assessed in > 350 individuals from four populations in North America. The loci had between 9 and 40 alleles and observed heterozygosities ranged from 0.071 to 0.87. Some of these pairs of primers also successfully amplified DNA from two other snake species.     

Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barley

 This study has analyzed the molecular basis and genetic behaviour of the polymorphism generated by the amplification of barley genomic DNA with primers complementary to microsatellites. Primers anchored at the 5′ end, used alone or in combination with arbitrary sequence primers, generated random amplified microsatellite polymorphisms (RAMPs). Unanchored primers were also used as single primers in a microsatellite primed-PCR (MP-PCR). Twenty six randomly selected RAMP DNA fragments which showed polymorphism between the cultivars Steptoe and Morex were cloned and sequenced. All sequences showed the expected repeated motif at the end of the insert, with the number of repeats ranging from five to ten. Genomic sequences containing low numbers of microsatellite motifs were preferentially amplified; therefore, only a fraction of the polymorphism could be attributed to variation in the number of microsatellite motifs at the priming site. Some sequences contained either cryptic simple sequences or members of families of repeated DNA. Polymorphism at the internal cryptic simple sequences was detected by RAMP bands inherited as co-dominant markers. Four MP-PCR bands were cloned and sequenced. A number of repeats identical to the primer itself were found at each end of the insert. Two allelic bands were polymorphic for an internal microsatellite. The potential use of cloned bands as fingerprinting tools was investigated by employing them as hybridization probes in Southern blots containing digested barley DNA from a sample of cultivars. RAMP probes produced complex hybridization band patterns. MP-PCR probes produced either a highly variable single locus or low-copy number loci. Segregations for 31 RAMPs and three MP-PCR bands were studied in a population of 70 doubled-haploids from the Steptoe/Morex cross. One third of all markers were co-dominantly inherited. Markers were positioned on an RFLP map and found to be distributed in all barley chromosomes. The new markers enlarged the overall length of the map to 1408 cM. Received: 6 May 1998 / Accepted: 20 July 1998     

微卫星位点筛选方法综述

微卫星标记因其丰富的多态性和共显性等特点,已得到了广泛的应用.应用微卫星标记首先需要获得微卫星位点的序列信息,用来设计引物.获得微卫星位点的方法有多种,本文综述了获得和富集微卫星位点的常用方法.最简便、最省时的方法是从公共数据库(如EMBL、Genbank、EST数据库等)或已发表的文献中查找到微卫星位点,但只限于已经有序列数据发布的物种.第二种方法是种间转移扩增,即从相近物种的数据库中查找微卫星位点,或使用已有数据发表的遗传距离相近物种的微卫星标记.第三种方法是从基因组DNA中筛选微卫星位点,其中用于富集微卫星的方法有引物法、磁珠杂交法、尼龙膜杂交法以及RAPD技术法.     

Automated screening and primer design of fish microsatellite DNA loci on pyrosequencing data

In recent years, massive sequencing approaches have allowed us to determine genomic structures of various organisms rapidly, raising novel applicability of the high-throughput sequence data obtained to various fields of biological studies. We present here a pipeline to search for microsatellite DNA and design PCR primers encompassing the microsatellites on genomic sequence data produced by 454 pyrosequencing. We tested this pipeline, called ‘Auto-primer’, on several fish genomic sequences and obtained many and various candidates for microsatellite DNA loci useful for detecting intraspecies genetic variability. This in silico search for microsatellite DNA is superior to conventional cloning methods, since any sequence patterns of repeat unit can be screened.     

One hundred fifty-four genetic markers for the turkey (Meleagris gallopavo).

Identifying and selectively breeding for improved traits is one of the ultimate goals of genetic research in agriculturally important species. Genome characterization and analysis are important first steps in this process. Genetic linkage maps based on the linear order of polymorphic DNA markers are typically developed through statistical analysis of inheritance patterns in pedigreed families. To develop microsatellite markers for further improvement of the turkey genetic linkage map, small-insert genomic libraries were screened for tandem repeats. Oligonuclotide primers were designed to amplify 164 microsatellite-containing fragments from genomic DNA. Genetic polymorphisms at 154 markers were determined by genotyping the F(1) individuals of two resource populations. Markers determined as segregating in the University of Minnesota/Nicholas Turkey Breeding Farms (UMN/NTBF) reference population were used to genotype F(2) individuals and a two-point linkage analysis was performed.