Rapid isolation of CA microsatellites from the tilapia genome

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.     

Fifty microsatellite markers for Japanese quail

A Japanese quail genomic library enriched for (CA/GT)n simple sequence repeats was screened and positive clones were sequenced. Fifty original microsatellite sequences were isolated that consisted mainly of perfect repeats of the dinucleotide (CA/GT)n motif and a corresponding number of polymerase chain reaction (PCR) primer pairs complementary to unique DNA sequences flanking the microsatellite repeats were designed to detect the repeats. Forty-six percent (23 of 50) of the markers revealed polymorphism in two unrelated quail individuals (one male and one female) randomly sampled from a population of wild quail origin. All 50 primer pairs were tested in the PCR for their ability to amplify chicken genomic DNA. Amplification products were obtained for 14 (28.0%) of the markers at the annealing temperature optimized for quail. These results provide an opportunity to begin characterizing the quail genome for the development of a genetic map for this economically valuable species and the eventual construction of a comparative genetic map in Phasianidae, which comprises a number of agriculturally important species of poultry.     

Isolation and characterization of 70 novel microsatellite markers from ostrich (Struthio camelus) genome.

Microsatellite markers are widely used in linkage mapping, parentage testing, population genetic studies, and molecular evolution studies in many agricultural species, while only a limited number of ostrich (Struthio camelus) microsatellites have been isolated. Thus, we constructed a random small-insert genomic library and a microsatellite-enriched library containing CA repeats. Fourteen clones containing CA repeats were isolated from 3462 clones in the non-enriched library by radioactive screening and 248 positive clones were isolated from 300 sequenced clones from the enriched library by PCR screening. After the enrichment procedures, the proportion of clones containing CA repeats was raised to 78.8%, compared with 0.4% in the non-enriched libraries, indicating that the enrichment value approaches 200 fold, which decreased the time and cost of cloning. The number of complete simple CA repeats in these positive clones ranged from 5 to 29. The primers for 94 of these microsatellites were developed and used to detect polymorphisms, of which 61 loci exhibited length polymorphisms in 17 unrelated ostrich individuals. The new polymorphic microsatellite markers we have identified and characterized will contribute to the ostrich genetic map, parentage testing, and comparative genomics between avian species.     

Polymorphic CA/GT and GA/CT microsatellite loci for Ostrinia nubilalis (Lepidoptera: Crambidae)

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short (n = 10–12). Ten microsatellites, PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range six to 20), and six of 10 loci showed heterozygote deficiency. Amplification of eight loci was observed in the sister species O. furnicalis.     

鳞翅目昆虫基因组中微卫星DNA的特征以及对其分离的影响

本文根据我们对鳞翅目昆虫棉铃虫和松毛虫以及其它动物 (筏蜘蛛、朱、鳕鱼和飞蝗 )的微卫星富集性基因组DNA文库的筛选和分析结果 ,结合其它实验室已发表的资料 ,对鳞翅目昆虫基因组中微卫星DNA的丰度和结构特点进行了较为系统的分析。结果表明 :与其它类群相比 ,尽管鳞翅目昆虫物种间存在差异 ,但其基因组中存在明显偏多的侧翼序列重复的、以多拷贝形式存在的微卫星位点 ,且其中相当一部分以基因家族的形式存在。微卫星DNA家族通常可以在序列分析阶段被识别出来 ,但很多多拷贝位点只有通过一系列后续分析才能被检查出来。这应是鳞翅目昆虫中微卫星位点的优化率相对偏低的主要原因。棉铃虫和松毛虫基因组中三相重复微卫星丰度相对较高 ,从而从某种程度上补偿了这些物种微卫星分离过程中因丰度低、多拷贝位点比例高所带来的困难。棉铃虫微卫星DNA家族侧翼序列中多聚T/A序列的存在表明 ,逆转录转座或逆转录侵染可能是在基因组中形成多拷贝微卫星位点和微卫星DNA家族的重要机制之一     

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)₁₁ probe for the presence of (CA/GT)_n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)_n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)_n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.)

 Ninety-four newly developed microsatellite markers were integrated into existing RFLP framework maps of four rice populations, including two doubled haploid, a recombinant inbred, and an interspecific backcross population. These simple sequence repeats (SSR) were predominantly poly(GA) motifs, targetted because of their abundance in rice. They were isolated from a previously described sheared library and a newly constructed enzyme-digested library. Differences in the average length of poly(GA) tracts were observed for clones isolated from the two libraries. The length of GA motifs averaged 21 repeat units for clones isolated from the Tsp-509-digested library, while motifs averaged 17 units for clones from the sheared library. There was no evidence of clustering of microsatellite markers near centromeres or telomeres. Mapping of the 94 newly developed markers as well as of 27 previously reported microsatellites provided genome-wide coverage of the 12 chromosomes, with an average distance of 1 SSLP (simple sequence repeat polymorphism) per 16–20 cM. Received: 13 February 1997/Accepted: 28 February 1997     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Abundance and DNA sequence of two-base repeat regions in tropical tree genomes

Tandem DNA repeats of two-base pairs are potentially important tools for population genetic studies because of their abundance and length variation. As part of our research into the ecology of tropical forest plants, we began a study of dinucleotide repeat regions in several genera of tropical trees. Genomic libraries in bacteriophage lambda were screened with the oligonucleotide probes poly(GT) and poly(AG). Both types of repeat regions were abundant in the genomes of all six plant species examined. Using the size of inserts in the phage libraries and number of phage screened, we estimated that there were 5 x 10(3) to 3 x 10(5) poly(AC) sites per genome, with slightly more AG than AC sites. When libraries were made from smaller fragments of genomic DNA, abundance estimates were higher, suggesting that two-base repeat sites were clustered in the genome. Poly(AC) sites were 16-22 bp in length, and four of the five sequenced were adjacent to either poly(AG)or poly(AT) sites. Other repeat region s appeared in DNA flanking the AC sites. This further demonstrated that two-base repeats and other repetitive DNA were clustered in the genome. Two-base repeats are abundant in plant genomes and could provide a large number of polymorphic markers for studies of plant population genetics.     

Isolation and sequencing of CA/GT repeat microsatellites from chromosomal libraries without subcloning.

A method for the isolation of (CA)n microsatellites from chromosome-specific genomic libraries is described. Clones were first screened using a polynucleotide CA/GT probe. Those shown to contain CA repeats were plaque purified and either subcloned or the insert amplified directly using vector primers. Polymerase chain reaction products were then used to directly sequence the regions flanking CA repeats by using biotinylated primers that amplify cloned inserts outward from CA repeat containing regions of DNA to vector primers. This method provides rapid access to microsatellites from chromosomes or chromosome regions of interest.     

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     

Silene tatarica microsatellites are frequently located in repetitive DNA

The genomic distribution of microsatellites can be explained by DNA slippage, slippage like processes and base substitutions. Nevertheless, microsatellites are also frequently associated with repetitive DNA, raising the question of the relative contributions of these processes to microsatellite genesis. We show that in Silene tatarica about 50% of the microsatellites isolated by an enrichment cloning protocol are associated with repetitive DNA. Based on the flanking sequences, we distinguished seven different classes of repetitive DNA. PCR primers designed for the flanking sequences of an individual clone amplified a heterogeneous family of repetitive DNA. Despite considerable variation in the flanking sequence (pi = 0.108), the microsatellite repeats did not show any evidence for decay. Rather, we observed the emergence of a new repeat type that probably arose by mutation and was spread by replication slippage. In fact, a complete repeat type switch could be observed among the analysed clones. We propose that the analysis of microsatellite sequences embedded in repetitive DNA provides a hitherto largely unexplored tool to study microsatellite evolution.     

黑斑狗鱼部分基因组文库构建和微卫星位点的筛选

采用磁珠富集与放射性杂交相结合的方法开发黑斑狗鱼(Esoxreieherti Dybowski)基因组微卫星资源。基因组DNA经Sau3AⅠ限制性内切酶消化后,选取400―900bp的片段进行PCR全基因组扩增,并利用生物素标记的(CA)12、(GA)12探针进行微卫星片段的富集。将得到的片段与pGEM-T载体连接后转入DH5α大肠杆菌中,然后利用γ-32P标记的放射性同位素探针进行第二次杂交。结果,共获得微卫星基因组文库1600个菌,杂交前菌落PCR检测阳性克隆率为90.91%;杂交后得到的阳性克隆为1300个,占87.25%。从中挑出196个进行测序,192(97.96%)个含有微卫星序列。在得到的微卫星序列中,重复单元除CA/GT、GA/CT外,还观察到单碱基、四碱基、五碱基重复单元。根据侧翼序列应用引物设计软件PrimerPremier5.0设计引物70对,选择合成32对,通过优化PCR反应条件,结果有28对引物可扩增出清晰可重复的目的条带。本研究旨在对黑斑狗鱼基因组资源的开发利用起到一定的促进作用,并为黑斑狗鱼养殖品系的优化、遗传多样性的检测及遗传图谱的构建等奠定基础。     

东方田鼠部分BAC文库的微卫星筛选

目的 从东方田鼠的部分BAC文库中筛选微卫星.方法 应用非放射性的菌落杂交方法和磁珠富集法从东方田鼠的BAC文库中筛选高质量的微卫星标记.结果 以地高辛标记的寡聚核苷酸(CA)20为探针,通过菌落杂交法从136个东方田鼠BAC克隆中筛选出杂交信号最强的20个阳性克隆.再将这20个阳性克隆分别通过链霉亲和素磁珠法构建亚克隆文库,从中选取400个经PCR鉴定为阳性的亚克隆进一步测序分析,共得到220个微卫星序列,阳性率55％.选取重复次数高,侧翼序列完整的微卫星序列设计74对引物,共有35对引物能扩增出清晰的条带,其中16对引物具有多态性.结论 成功且高效地从阳性BAC克隆中筛选出微卫星序列,这些微卫星和阳性BAC克隆可用于后续的定位研究.     

Rapid identification of polymorphic CA-repeats in YAC clones

Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci. The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.