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Genome Fingerprinting by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction Amplification
Abstract
Simple sequence repeats (SSR), or microsatellites, are ubiquitous in eukaryotic genomes. Here we demonstrate the utility of microsatellite-directed DNA fingerprinting by polymerase chain reaction (PCR) amplification of the interrepeat region. No sequencing is required to design the oligonucleotide primers. We tested primers anchored at 3' or 5' termini of the (CA)n repeats, extended into the flanking sequence by 2 to 4 nucleotide residues [3'-anchored primers: (CA)8RG, (CA)8RY, and (CA)7RTCY; and 5'-anchored primers: BDB(CA)7C, DBDA(CA)7, VHVG(TG)7 and HVH(TG)7T]. Radioactively labeled amplification products were analyzed by electrophoresis, revealing information on multiple genomic loci in a single gel lane. Complex, species-specific patterns were obtained from a variety of eukaryotic taxa. Intraspecies polymorphisms were also observed and shown to segregate as Mendelian markers. Inter-SSR PCR provides a novel fingerprinting approach applicable for taxonomic and phylogenetic comparisons and as a mapping tool in a wide range of organisms. This application of (CA)n repeats may be extended to different microsatellites and other common dispersed elements.
... In this study we used intersimple sequence repeat (ISSR) 1 BD 5-27 ------Israel 2 BD 17-70 ------Israel 3 Bedana 11-93 India India 4 Bengal 3-75 Florida India 5 Bengali 1-39 India India 6 Black Leaf y 9-85 Florida China Hei Ye 7 Bombai 17-93 India India 8 Brewster 38 markers which are generated by microsatellite-repeat-anchored primers amplifying genomic segments between adjacent SSR loci (Zietkiewicz et al., 1994). These primers target microsatellites that are abundant throughout the plant genome (Wang et al., 1994), making prior knowledge of the target sequences unnecessary. ...
... These primers target microsatellites that are abundant throughout the plant genome (Wang et al., 1994), making prior knowledge of the target sequences unnecessary. These primers offer great potential for fingerprinting compared to other arbitrary primers, since they simultaneously target multiple genomic loci (Zietkiewicz et al., 1994). Polymorphisms occur whenever one genome lacks the repeated sequence or has a deletion or insertion that modifies the distance between repeats. ...
... Polymorphisms occur whenever one genome lacks the repeated sequence or has a deletion or insertion that modifies the distance between repeats. The ISSR method can differentiate between closely related individuals (Fang and Roose, 1997;Moreno et al., 1998;Zietkiewicz et al., 1994). ISSR markers have been used for cultivar identification and for genetic relationship studies in various plant species (Ajibade et al., 2000;Blair et al., 1999;Fang and Roose, 1997;Fang et al., 1998;Gilbert et al., 1999;Goulao et al., 2001;Lanham and Brennan, 1998;Martin and Sanchez-Yelamo, 2000;Moreno et al., 1998;Prevost and Wilkinson, 1999). ...
There is widespread confusion and uncertainty concerning the identity of lychee cultivars: the same cultivar may be known under different names and different cultivars may appear under the same name. In the present study, the potential of intersimple sequence repeat (ISSR) for the identification of 66 lychee cultivars and accessions and a determination of their genetic relationships was evaluated, using 32 primers containing different simple sequence repeat motifs. Of the 194 bands produced, 124 (64%) were polymorphic. A set of six ISSR primers was sufficient to distinguish all cultivars and accessions. Thus, cultivars which are morphologically very similar and have identical isozyme profiles can be distinguished by ISSR analysis. However, seven pairs of accessions, each considered to be the same cultivar, were found to be identical by ISSR analysis. Nei and Li band-sharing distances and Nei genetic distances were calculated among the cultivars and two similarity dendrograms were generated using the neighbor-joining algorithm. Results showed that the ISSR technique is a valuable tool for identification of lychee cultivars and analysis of their genetic relationships.
... Although various types of markers have been developed, the inter-simple sequence repeat (ISSR) is very suitable for this use. ISSR markers use degenerate primers to reveal a large number of fragments per PCR reaction, and thus are able to efficiently distinguish between closely related individuals (Zietkiewicz et al., 1994). In citrus, ISSR markers are well distributed over linkage groups Sankar and Moore, 2001) and there is little tendency for linkage between markers amplified with a single degenerate primer. ...
... Each reaction mixture was overlaid with 50 µL of mineral oil. Amplification products were separated on 320 × 380 × 0.4 mm 6% nondenaturing polyacrylamide gels containing 3 M urea and 1× Tris-borate/EDTA (TBE) buffer at 450 V. (Zietkiewicz et al., 1994). All other seed lots were amplified in a Stratagene Robocycler (La Jolla, Calif.) using 15 µL reactions and a program of 3 min at 94 °C, then 35 cycles of 1 min at 94 °C, 1 min at 52°C, and 2.5 min at 72 °C, then a final extension for 7 min at 72 °C. ...
New potential citrus germplasm accessions may be received as seed rather than budwood, thereby reducing phytosanitary risks. However, trueness-to-type may be an issue with seed materials because many varieties produce both apomictic (nucellar) and sexual (zygotic) embryos and most citrus is fairly heterozygous. To identify nucellar seedlings of polyembryonic types and to retain these as representing the type, we screened 1340 seedlings from 88 seed sources for markers amplified with two inter-simple sequence repeat (ISSR) primers. Sixteen seed sources produced no seedlings classified as being of nucellar origin. Among the remaining seed sources, seedlings classed as nucellar were identified for potential addition to the collection. In 37 accessions, both nucellar and zygotic seedlings were detected, and in some cases both types were retained. Inclusion of established accessions of the same cultivar group in the analysis allowed an initial assessment of similarity to existing accessions. This technique improved the efficiency of acquiring new germplasm of polyembryonic types by seed. The method identifies those seed sources that produce few or no nucellar seedlings, but it is not useful for determining which seedlings of monoembryonic types should be retained in collections.
... Геномне сканування набуває особливого значення в дослідженнях генофондів тварин, оскільки тільки наявність достатньої кількості геномних молекулярно-генетичних маркерів дозволяє зберігати, контролювати і удосконалювати необхідне біорізноманіття таких видів, сканування може проводитися як на рівні декількох десятків або сотень маркерів, так і в об'ємі повного секвенування геномів (Lowry, 2010). На сьогодні розроблено достатньо багато методів подібного сканування, до одного з них відноситься геномне полілокусне генотипування за фрагментами ДНК різної довжини, франкованих інвертованим повтором мікроса-телітних локусів (Inter-SimpleSequenceRepeat -ISSR-PCR маркери) (Zietkiewicz et al., 1994). Інвертовані повтори викликають особливу цікавість, оскільки схильність до формування вторинних структур ДНК складає основу для геномної нестабільності в ділянках їх локалізації (Cook et al., 2011). ...
The aim of this study is to analyze the informativeness of ISSR markers in the study of intrapopulation genetic polymorphism of the Lebedyn local breed cattle population. Materials and methods of research. To confirm the uniqueness and consolidation of the Lebedyn breed cow population, an assessment of the genetic polymorphism of the studied cows was carried out using molecular diagnostics (ISSR-fingerprinting). The material for assessing intrapopulation genetic variability was blood samples taken from cows of the local Lebedyn breed (n = 33 heads), which are kept in the Boryspil district of the Kyiv region in the Holosiyivske farm, Ukraine. Molecular genetic studies were carried out on the basis of the аnimal genetics laboratory of the M.V. Zubets Institute of Animal Breeding and Genetics of the National Academy of Sciences of the Ukraine. Results. The genetic structure of the population of the local small-sized Lebedyn cattle breed of Ukraine was analyzed using 8 ISSR systems based on the following microsatellites: (ACC)6G, (CTC)6C, (GAG)6C, (GA)6CC, (AG)8CG, (AG)8CA, (GA)9C and (AG)9C. The spectra of amplification products using different microsatellite loci as primers annealing sites and amplification of sites located between their inverted repeats significantly differed from each other. The difference was both in the number of amplicons obtained, their length (in nucleotide pairs), and their polymorphism. 88 amplified DNA fragments were identified, of which only 18 were polymorphic. The total number of polymorphic loci was 20.45%. According to the results of population genetic analysis, the (AG)8CGmarker was the least polymorphic (PIC = 0.115). Conclusion. These results indicate a high degree of genetic consolidation and possible reproductive isolation of the population of the studied animals.
... Inter-simple sequence repeat (ISSR) are reliable markers that do not require prior knowledge of the target sequences and are highly reproducible due to the length of the primers and the high annealing temperature. Additionally, these markers were found to be highly polymorphic (Zietkiewicz et al., 1994;Kojima et al., 1998;INTRODUCTION Bornet & Branchard, 2001). ISSR markers were widely used to study the genetic diversity of insects including species of leafhoppers (Hemiptera: Cicadellidae) in the USA (de León et al., 2004) and rice planthopper (Hemiptera: Delphacidae) pests in Asia (Liu et al., 2010;Xie et al., 2014;Nam et al., 2019;Babu et al., 2023). ...
The corn leafhopper, Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) is the vector of the Corn stunt disease, caused by a complex of pathogens, a serious threat to corn production in tropical and subtropical regions of the American continent. The aim of this study was to conduct an exploratory analysis to estimate the structure and genetic differentiation of D. maidis populations from three geographical regions collected during two maize growing seasons in northwestern Argentina using inter-simple sequence repeats (ISSR) markers. Populations in the High-elevation Valley and Dry Plain sites, despite being geographically separated by mountains of high elevations, were genetically similar, while the nearest populations located in Dry Plain and Humid Piedmont were the most genetically different. The results suggested that populations of D. maidis in subtropical maize-growing areas are structured according to the climatic conditions, mainly humidity and precipitation. Additionally, this structure might be influenced by annual migrants from northern tropical areas, as well as by individuals that occasionally survive from one season to the next as occurs in the Humid Piedmont. Darker body coloration of females sampled during the second year in Dry Plain and Humid Piedmont might be associated with colder temperatures during the winter before maize cultivation. This study is the first conducted in Argentina on the genetic diversity of populations of D. maidis. The results presented here highlight the need for further investigation into the origin and potential movement of D. maidis populations. Knowing how the crop is colonized will aid in the development of well-fitting models, for preventing the disease caused by the pathogens transmitted by this insect.
... Spectrophotometry and horizontal agarose gel electrophoresis on a 0.8% agarose gel were used to ensure sufficient quantity and quality of genomic DNA. The SSR markers were used as anchors in constructing a genetic map 26 . In addition, Inter Simple Sequence Repeat (ISSR) 27 , Random Amplified Polymorphic DNA (RAPD) 28 , interprimer binding site (iPBS) 29 , inter retrotransposon amplified polymorphism (IRAP) 30 6% polyacrylamide gel was used to separate the layers, and gel staining was performed using the silver nitrate method 33 . ...
By employing machine-learning models, this study utilizes agronomical and molecular features to predict powdery mildew disease resistance in Barley (Hordeum Vulgare L). A 130-line F8-F9 barley population caused Badia and Kavir to grow at the Gonbad Kavous University Research Farm on three planting dates (19 November, 19 January, and 19 March), with three replicates in 2018/2019 and 2019/2020. The study employed RReliefF, MRMR, and F-Test feature selection algorithms to identify essential phenotype traits and molecular markers. Subsequently, Decision Tree, Random Forest, Neural Network, and Gaussian Process Regression models were compared using MAE, RMSE, and R2 metrics. The Bayesian algorithm was utilized to optimize the parameters of the machine-learning models. The results indicated that the Neural Network model accurately predicted powdery mildew disease resistance in barley lines. The evaluation based on high R2 values, as well as low MAE and RMSE, highlighted the efficacy of these models in identifying significant phenotype traits and molecular markers associated with disease resistance. The findings demonstrate machine learning models’ potential in accurately predicting powdery mildew disease resistance in Barley. The neural network model specifically showed excellent results in this area because it managed to identify critical phenotypic traits and molecular markers very well. This research highlights the importance of combining AI with molecular markers for improved disease resistance and other desirable crop traits during plant breeding.
... Amplified Fragment Length Polymorphism (AFLP) combined the advantages of RFLP and PCR, facilitating the generation of numerous markers without requiring prior sequence information [17]. With the advancement of technology, Inter-Simple Sequence Repeat (ISSR) markers, which exploit variations in microsatellite amplification, have provided enhanced resolution [18]. SNP markers, widely distributed and genetically stable, have become one of the most widely used molecular markers in recent years [19]. ...
Eggplant (Solanum melongena) is a significant vegetable in the Solanaceae family. Significant progress has been made in genetic diversity analysis and fingerprinting construction for crops such as tomatoes and peppers within the same family, but research on eggplants in these aspects remains relatively limited. Current germplasm identification using fingerprinting primarily relies on traditional SSR markers, which suffer from limited polymorphism and labor-intensive workflows. This study aimed to identify high-quality single nucleotide polymorphisms (SNPs), develop reliable Kompetitive Allele-Specific PCR (KASP) markers for eggplant genotyping, and then conduct fingerprint construction and genetic diversity analysis. The ultimate goals were to achieve a precise identification of eggplant varieties and deeply explore the genetic background and evolutionary patterns of eggplant germplasm. In this study, 49 representative eggplant accessions were re-sequenced. After data quality control, sequence alignment, and multiple rounds of screening, 224 high-quality SNPs were identified. Based on these SNPs, 96 SNPs were selected to develop KASP markers. These markers can provide abundant genetic markers for eggplant genetic research, which are used to deeply explore the genetic background and conduct genetic diversity analysis. After multiple rounds of rigorous verification, 32 core candidate markers were finally screened out. The average polymorphic information content (PIC) and gene diversity (GD) values were 0.36 and 0.46, respectively. Phylogenetic tree, population structure, and principal component analyses divided the 280 eggplant accessions into eight distinct groups. Through the analysis of minimal core markers and core germplasm, 23 core SNP markers and a subset of 56 core germplasm accessions were identified, leading to the establishment of a comprehensive fingerprinting system for all 280 accessions. Our findings provide a foundational genetic resource for eggplant germplasm identification and offer significant support for future breeding efforts.
... They can cover the entire genome with a large number of markers exhibiting high polymorphism [17]. The main molecular marker technologies include Restriction Fragment Length Polymorphism (RFLP) [18], Random Amplified Polymorphic DNA (RAPD) [19], Inter-Simple Sequence Repeat Polymorphism (ISSR) [20], Single-Nucleotide Polymorphism (SNP) [21], Amplified Fragment Length Polymorphism (AFLP) [22], and simple sequence repeats (SSRs) [23]. Microsatellite markers, also known as short tandem repeats or simple sequence repeats, are simple repetitive sequences that are uniformly distributed in the genomes of eukaryotes. ...
Alfalfa, as a high-quality forage resource, has high nutritional value. Due to the high phenotypic similarity among its varieties and the susceptibility to environmental influences, challenges are encountered in variety identification and breeding. In this study, 23 simple sequence repeat (SSR) markers were screened to distinguish 49 alfalfa varieties, among which 21 SSR markers showed polymorphic fragments. The results indicated that these 21 markers were highly polymorphic, with an average of 5.91 alleles per SSR marker locus and an average polymorphic information content (PIC) of 0.66, suggesting a strong discriminatory efficiency. The results of a population genetic diversity analysis showed that there was a relatively high level of genetic diversity among the tested materials. The analysis of molecular variance (AMOVA) results indicated that the genetic variation within the population of the 49 alfalfa germplasm samples was the main source of the total variation. The results of genetic distance and genetic identity analyses showed that the genetic relationship between population 1 and population 4 was the most distant, while the relationship between population 2 and population 3 was the closest. The cluster analysis results showed that samples S16 and S55 formed a separate branch; that is, there were two main genetic subgroups. These results confirm that SSR markers are effective tools for genetic characterization and precise discrimination of alfalfa varieties and have important application values in breeding, variety registration, and germplasm resource conservation.
Molecular marker breeding is a powerful tool for screening targeted genetic material, evaluating genetic differences and targeting a crop improvement potential. Hence, DNAs of leaf samples belonging to 87 Rubus genotypes from different regions of Türkiye were isolated with the application marking system. Polymerase chain reaction (PCR) products were obtained with 16 inter simple sequence repeat (ISSR) primers and band lengths ranging from 150 bp [(AGC)6G, (AG)7YC, VHVG(TG)7] to 1500 bp [HVH(CA)7 T] were detected. A total of 293 bands were obtained in terms of genetic diversity and 292 were shown to be polymorphic and 99.75% polymorphism rate. When the obtained results were evaluated, it was confirmed that there was a significant genetic diversity in the genetic material. In the molecularly presented dendrogram, the similarity level between genetic materials can be determined between 0.68 and 1, the average similarity coefficient was determined as 0.84 and two main groups (A and B) were established. Almost all of the genotypes are genetically separated from each other.
Rubus cultivars and genotypes were divided into five populations using a model-based structure algorithm. Based on the STRUCTURE analysis, individuals in the second, fourth, and fifth subpopulations were identified as pure cultivars and are expected to contribute to hybridization research. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis and principal component analysis (PCA) also supported these five different populations and confirmed the reliability of the STRUCTURE analysis. According to the obtained data, the low number of populations was due to the high gene flow rate between the regions where the samples were taken.
The study is the first in this field with blackberry materials representing different regions of Türkiye. The genetic diversity observed in the Rubus genetic material reveals that this material is a valuable genetic resource for comprehensive breeding programs. In addition, these cultivars and genotypes are considered as promising candidates for breeding aimed at increasing blackberry production.
The pawpaw [ Asimina triloba (L.) Dunal.] is a tree fruit native to many areas of the southeastern and mid-western United States. Kentucky State University (KSU) is designated as a satellite repository for Asimina for the U.S. Department of Agriculture (USDA), National Plant Germplasm System (NPGS). An assessment of the level of genetic diversity in cultivated pawpaw would assist in development of the future germplasm repository collection strategies for cultivar improvement. The objectives of this study were to identify intersimple sequence repeat (ISSR) markers that segregate in a simple Mendelian fashion and to use these markers to assess genetic diversity in 19 pawpaw cultivars. Leaf samples from the 34 progeny of controlled crosses (1-7-1 × 2-54 and reciprocal) and the parents were collected, DNA was extracted, and subjected to the ISSR methodology using the University of British Columbia microsatellite primer set #9. Seven primers yielded 11 Mendelian markers with either a 3:1 or 1:1 ratio that was confirmed by chi-square analysis. Analysis of genetic diversity using 10 of the ISSR markers from 19 pawpaw cultivars revealed a moderate to high level of genetic diversity, with a percent polymorphic loci P = 80 and an expected heterozygosity He = 0.358. These diversity values are higher than those reported for cultivated pawpaw using isozyme or randomly amplified polymorphic DNA (RAPD) markers, indicating that the ISSR marker methodolgy has a higher level of discrimination in evaluating genetic diversity in pawpaw and/or pawpaw has greater levels of genetic diversity than previously found.
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We present the use of our recently described multiple-loci polymorphic DNA markers ("alumorphs") for linkage mapping of the human genome. By using the polymerase chain reaction (PCR) with an Alu-specific primer we could reveal, in a single experiment, up to 20 genomic polymorphisms seen as the presence or absence of amplified DNA fragments originating from genomic segments flanked by Alu repeats. Using this approach we examined genomic DNA samples from two families with a history of pseudovitamin D-deficiency rickets (PDDR), an autosomal recessive disorder. An indication of linkage with the PDDR phenotype was found for one of the polymorphic bands, denoted 30A. A significant linkage [logarithm-of-odds (lod) score greater than 3.0] was obtained between this polymorphism and a number of chromosome 12q markers tightly linked to PDDR. The 30A band specifically hybridized to DNA digests from hybrid cell lines carrying a human chromosome 12, thus independently assigning the 30A marker to this chromosome. Since Alu elements are ubiquitous in human DNA, the use of alternative Alu-specific primers, which reveal different sets of Alu-flanked loci, should provide an efficient and rapid approach to human genetic mapping.
Methods for the production and use of human DNA fragments using the polymerase chain reaction and oligonucleotide primers directed to repetitive sequences are described. Primers using the human Alu and L1Hs repeat elements, including a number of different Alu primers, are delineated and compared. Interpretation of patterns of amplification in somatic cell hybrids by ethidium bromide staining and hybridization analysis is discussed, with particular attention to use of repeat-containing DNA fragments as hybridization probes. The method can also be applied to DNAs cloned into Escherichia coli and yeast vector systems, and primer sequences from yeast artificial chromosome, λ, and cosmid vectors useful for these experiments are provided.
A method was recently developed for the specific amplification of human DNA sequences from interspecific somatic cell hybrids by the polymerase chain reaction (PCR) using primers directed to Alu, a short interspersed repeat element (SINE). We now show human-specific amplification using a primer to the 3′ end of the human long interspersed repeat element L1Hs (LINE). A monochromosomal hybrid containing an intact human X chromosome yielded approximately 25 discrete products, ranging in size from 800 to 4500 bp. Combination of a single Alu primer and the L1Hs primer yielded a large number of smaller products (300–1000 bp) distinct from those observed with either primer alone. Inspection of ethidium bromidestained gels showed one Alu-Alu and three Alu-L1Hs products which were present in an intact X chromosome but absent in a hybrid containing an X chromosome deleted for the single metaphase band q28. These four fragments were isolated from the gel and used as probes on Southern blots which confirmed their localization to Xq28. These results demonstrate that primers can be constructed to a variety of interspersed repetitive sequences (IRS) and used individually or in combination for the rapid isolation of DNA fragments from defined chromosomal regions by IRS-PCR.
The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.
During a search of polymorphic microsatellites for bovine genome mapping, we found that microsatellites often occur as tails
of artiodactyl C-A retroposon elements. In this element, C (85bp) is a tRNA derivative, while A (117bp) is of unknown origin.
The A element also occurs as dimer element with a connecting 27bp linker sequence comprising hexanucleotide CACTTT repeats.
In 10 clones (45% of those selected deliberately for dinucleotide repeats), the microsatellite motif is associated with the
C-A retroposon. In 50% of 44 database artiodactyl C-A sequences, the element also has a microsatellite tail. The microsatellite
is usually a simple (CA)n repeat, but in some cases it is an apparent derivative of the linker sequence CACTTT. All but one of 33 database dimer elements
have trinucleotide repeat tails (AGC)n, n = 1–9. Microsatellites, retroposons, and their truncated versions (C and/or A) often occur as clusters. We derived the
consensus sequence (202bp) of the C-A element, and designed four primers for inter-SINE amplification with the aim of finding
SINEmorph polymorphisms. The method is potentially powerful for rapidly producing polymorphic markers for artiodactyl genome
mapping.
A linkage map of the human genome has been constructed based on the segregation analysis of 814 newly characterized polymorphic loci containing short tracts of (C-A)n repeats in a panel of DNAs from eight large families. Statistical linkage analysis placed 813 of the markers into 23 linkage groups corresponding to the 22 autosomes and the X chromosome; 605 show a heterozygosity above 0.7 and 553 could be ordered with odds ratios above 1,000:1. The distance spanned corresponds to approximately 90% of the estimated length of the human genome.
Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg2+, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.
The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.
Length polymorphisms within simple-sequence loci occur ubiquitously in non-coding eukaryotic DNA and can be highly informative in the analysis of natural populations. Simple-sequence length polymorphisms (SSLP) in the long-finned pilot whale Globicephala melas (Delphinidae) have provided useful information on the mating system as well as on the genetic structure of populations. We have therefore tested whether the polymerase chain reaction primers designed for Globicephala could also be used to uncover variability in other whale species. Homologous loci could indeed be amplified from a diverse range of whales, including all toothed (Odontoceti) and baleen whales (Mysticeti) tested. Cloning and sequencing these loci from 11 different species revealed an unusually high conservation of sequences flanking the simple-sequence stretches, averaging 3.2% difference over 35-40 Myr. This represents the lowest divergence rate for neutral nucleotide positions found for any species group so far and raises the possible need for a re-evaluation of the age of the modern whales. On the other hand, the high conservation of non-coding sequences in whales simplifies the application of SSLP DNA fingerprinting in cetacean species, as primers designed for one species will often uncover variability in other species.
The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Alu repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Alu repeats, these markers, called "alumorphs," could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found.