Large Number Of Microsatellite Markers Research Articles

Genome survey sequencing and mining of genome-wide microsatellite markers in yellow-billed babbler (Turdoides affinis)

Turdoides affinis is a species of group dwelling old world passerine of family Leiothrichidae. Unavailability of genome-wide sequence and species-specific molecular markers have hindered comprehensive understanding of cooperative breeding behaviour in T. affinis. Therefore, we generated genome-wide microsatellite markers through whole genome short read sequencing of T. affinis. A total of 68.8 gigabytes of paired-end raw data were sequenced containing 195,067,054 reads. Total sequenced reads spanned a coverage of 17X with genome size of 1.18 Gb. A large number of microsatellite markers (265,297) were mined in the T. affinis genome using Krait, and 50 most informative markers were identified and validated further. In-silico PCR results validated 47 markers. Of these 47 markers, five were randomly selected and validated in-vitro in twelve individuals of T. affinis. Genotyping data on these five loci estimated observed heterozygosity (H0) and expected heterozygosity (He) ratios between 0.333 – 0.833 and 0.851–0.906, respectively. Effective allele size ranged from 6.698 to 10.667, inbreeding coefficient of the population ranged from 0.080 to 0.631 and null allele frequency was calculated at 0.055 to 0.303. Polymorphic information content of all the five loci varied between 0.850 and 0.906. Probabilities of exclusion and identity across 5 loci was estimated to be 0.95 and 0.0036, respectively. All the loci showed significant adherence to Hardy-Weinberg equilibrium. The microsatellite markers reported in this study will facilitate future population genetics studies on T. affinis and other congeneric species.

Genetic diversity of an invasive earthworm, Lumbricus terrestris, at a long-term trading crossroad, the Champlain Valley of Vermont, USA

The invasive European earthworm, Lumbricus terrestris, is now widely distributed in North America. This success may result from high genetic diversity derived from multiple introductions from founder sources across Europe. Using a mitochondrial gene (COI) and microsatellite markers, L. terrestris from seven sites in the Champlain Valley of Vermont USA were scored for genetic diversity and population structure. This region has been a trading crossroads for centuries, thus likely to have received earthworms from multiple origins. COI sequences matched those reported for L. terrestris from Scotland, France, Austria, Denmark, Sweden, and Norway, and 2–5 haplotypes were found at each site. Genetic diversity (microsatellites) was great for each site, but not notably greater than for earthworm populations in general, possibly because there may be allele size homoplasy, or some restriction in the number of alleles possible at any locus. The earthworms were genetically differentiated among the Vermont study sites, even those 0.6–13 km distant. These results support the view that L. terrestris is a successful invasive earthworm because multiple introductions provided ample genetic variation for natural selection and local differentiation among locations in North America. Last, a large number of microsatellite markers is provided, including suggested PCR programs, for free use by future researchers.

Development and Characterization of SSR Markers to Study Genetic Diversity and Population Structure of Horsegram Germplasm (Macrotyloma uniflorum)

Development of genomic resources in any crop is the pre-requisite for the construction of linkage map and implementation of molecular breeding strategies to develop superior cultivars. Large number of molecular markers are required to enrich the scanty information available in horsegram (Macrotyloma uniflorum).We employed the next-generation Illumina sequencing platform to develop a large number of microsatellite markers in this species. Of the total 23,305 potential SSRs motifs, 5755 primers were designed. Of these, 1425, 1310, 856, 1276, and 888 were of di-, tri-, tetra-, penta-, and hexa-nucleotide repeats respectively. Thirty polymorphic SSR primers and 24 morphological traits were used in 360 horsegram accessions to detect the genetic diversity and population structure. Thirty primers amplified 170 polymorphic alleles with an average of 5.6 alleles per primer having size 80 to 380 bp. The polymorphism information content (PIC) ranged from 0.15 to 0.76 with an average of 0.50, suggesting that SSR markers used in the study were polymorphic and suitable for characterization of horsegram germplasm. Dendrogram-based on Jaccard’s similarity coefficient and neighbor-joining tree grouped the horsegram accessions into two major clusters. Similarly, STRUCTURE analysis assigned genotypes into two gene pools namely Himalayan origin and Southern India. Diversity analysis based on 24 agro-morphological traits also suggested the presence of high level of diversity among the accessions.

Genome-Wide Identification and Transferability of Microsatellite Markers between Palmae Species.

The Palmae family contains 202 genera and approximately 2800 species. Except for Elaeis guineensis and Phoenix dactylifera, almost no genetic and genomic information is available for Palmae species. Therefore, this is an obstacle to the conservation and genetic assessment of Palmae species, especially those that are currently endangered. The study was performed to develop a large number of microsatellite markers which can be used for genetic analysis in different Palmae species. Based on the assembled genome of E. guineensis and P. dactylifera, a total of 814 383 and 371 629 microsatellites were identified. Among these microsatellites identified in E. guineensis, 734 509 primer pairs could be designed from the flanking sequences of these microsatellites. The majority (618 762) of these designed primer pairs had in silico products in the genome of E. guineensis. These 618 762 primer pairs were subsequently used to in silico amplify the genome of P. dactylifera. A total of 7 265 conserved microsatellites were identified between E. guineensis and P. dactylifera. One hundred and thirty-five primer pairs flanking the conserved SSRs were stochastically selected and validated to have high cross-genera transferability, varying from 16.7 to 93.3% with an average of 73.7%. These genome-wide conserved microsatellite markers will provide a useful tool for genetic assessment and conservation of different Palmae species in the future.

A first generation microsatellite-based linkage map of the Chinese mitten crab Eriocheir sinensis and its application in quantitative trait loci (QTL) detection

Genetic linkage maps provide excellent tools for detecting quantitative trait loci (QTL) and understanding genome organization and evolution. By taking advantage of a large number of microsatellite markers isolated from the genome of the Chinese mitten crab Eriocheir sinensis in our previous study, here we constructed a first generation genetic linkage map for this species using 1300 microsatellite markers that genotyped a F1 full-sib family from an intercross between northern and southern populations in China. A total of 674 markers (52%) were informative and grouped on the two parental maps. The number of linkage groups is quite close to the actual number of the crab haploid chromosomes (n=73). The female linkage map contained 466 markers which mapped to 71 linkage groups, spanning a total genetic distance of 2430.9cM with an average marker space of 6.2cmd The male map contained 457 markers which mapped to 70 linkage groups, spanning 2658.4cM with an average marker space of 6.9cmd The crab genome length was estimated to be 3390.0cM, covering 71.7% of genome for the female map, and 3678.8cM covering 72.3% of genome for the male map. Furthermore, the linkage maps were used for preliminary detection of QTL. Nine growth-related QTL for body length, width and weight were mapped to seven linkage groups. Additionally, two QTL were identified to be associated with sexual precocity. The linkage maps and the identified QTL will be valuable for marker-assisted selection breeding programs of the mitten crab. Statement of relevanceA first generation genetic linkage map of the Chinese mitten crab was created using 1300 microsatellite markers. Nine growth-related QTL and two sexual precocity-related QTL were identified, which will be valuable for further studies of the genetic mechanisms of development and sexual precocity, and marker-assisted selection breeding programs for this species.

Development and Integration of Genome-Wide Polymorphic Microsatellite Markers onto a Reference Linkage Map for Constructing a High-Density Genetic Map of Chickpea.

The identification of informative in silico polymorphic genomic and genic microsatellite markers by comparing the genome and transcriptome sequences of crop genotypes is a rapid, cost-effective and non-laborious approach for large-scale marker validation and genotyping applications, including construction of high-density genetic maps. We designed 1494 markers, including 1016 genomic and 478 transcript-derived microsatellite markers showing in-silico fragment length polymorphism between two parental genotypes (Cicer arietinum ICC4958 and C. reticulatum PI489777) of an inter-specific reference mapping population. High amplification efficiency (87%), experimental validation success rate (81%) and polymorphic potential (55%) of these microsatellite markers suggest their effective use in various applications of chickpea genetics and breeding. Intra-specific polymorphic potential (48%) detected by microsatellite markers in 22 desi and kabuli chickpea genotypes was lower than inter-specific polymorphic potential (59%). An advanced, high-density, integrated and inter-specific chickpea genetic map (ICC4958 x PI489777) having 1697 map positions spanning 1061.16 cM with an average inter-marker distance of 0.625 cM was constructed by assigning 634 novel informative transcript-derived and genomic microsatellite markers on eight linkage groups (LGs) of our prior documented, 1063 marker-based genetic map. The constructed genome map identified 88, including four major (7–23 cM) longest high-resolution genomic regions on LGs 3, 5 and 8, where the maximum number of novel genomic and genic microsatellite markers were specifically clustered within 1 cM genetic distance. It was for the first time in chickpea that in silico FLP analysis at genome-wide level was carried out and such a large number of microsatellite markers were identified, experimentally validated and further used in genetic mapping. To best of our knowledge, in the presently constructed genetic map, we mapped highest number of new sequence-based robust microsatellite markers (634) which is an advancement over the previously documented (~300 markers) inter-specific genetic maps. This advanced high-density map will serve as a foundation for large-scale marker validation and genotyping applications, including identification and targeted mapping of trait-specific genes/QTLs (quantitative trait loci) with sub-optimal use of resources and labour in chickpea.

Genomic-derived microsatellite markers for diversity analysis in Jatropha curcas

New set of 1,122 novel genomic-derived microsatellite markers developed, validated and a set of 447 polymorphic SSR identified for Jatropha curcas. The AMOVA showed that 6 % of the total genetic variation exists among the population and 94 % within the populations. Jatropha curcas L., a non-edible, oil-rich crop has attracted global attention as a promising renewable resource for biodiesel production. Limited numbers of validated and characterized markers such as SSRs are available that could be utilized for molecular breeding in Jatropha. To enrich the marker resources, we have employed next-generation 454 sequencing to develop a large number of microsatellite markers from trinucleotide-enriched genomic libraries. A total of 124,279 raw reads were assembled into 2,845 contigs and 22,650 singletons. We designed 1,122 SSR flanking primer pairs and validated them with a panel of seven accessions of J. curcas and one J. integerrima to identify polymorphic SSRs. Out of these 1,122 SSRs, 447 (39.83 %) were found to be polymorphic among J. curcas accessions. A higher transferability of SSRs (76 %) to J. integerrima was noticed. A subset of randomly selected SSRs (41 SSRs) was amplified across 96 accessions of J. curcas, which detected a total of 152 alleles ranging from 2 to 9 with an average of 4.0 ± 1.9 alleles/SSR. The analysis of molecular variance (AMOVA) showed that 6 % of the total genetic variation exists among the population and 94 % within the populations indicated high genetic variation within population. The neighbor-joining (NJ) tree based on Jaccard’s coefficient grouped the 96 accessions into three major clusters. The non-toxic accessions form a separate cluster and rest of the accessions intermixed irrespective of geographical distribution. STRUCTURE-based clustering also resulted in a similar pattern of grouping.

A microsatellite genetic linkage map of black rockfish (Sebastes schlegeli)

Ovoviviparous black rockfish (Sebastes schlegeli) is an important marine fish species for aquaculture and fisheries in China. Genetic information of this species is scarce because of the lack of microsatellite markers. In this study, a large number of microsatellite markers of black rockfish were isolated by constructing microsatellite-enriched libraries. Female- and male-specific genetic linkage maps were constructed using 435 microsatellite markers genotyped in a full-sib family of the fish species. The female linkage map contained 140 microsatellite markers, in which 23 linkage groups had a total genetic length of 1334.1 cM and average inter-marker space of 13.3 cM. The male linkage map contained 156 microsatellite markers, in which 25 linkage groups had a total genetic length of 1359.6 cM and average inter-marker distance of 12.4 cM. The genome coverage of the female and male linkage maps was 68.6% and 69.3%, respectively. The female-to-male ratio of the recombination rate was approximately 1.07:1 in adjacent microsatellite markers. This paper presents the first genetic linkage map of microsatellites in black rockfish. The collection of polymorphic markers and sex-specific linkage maps of black rockfish could be useful for further investigations on parental assignment, population genetics, quantitative trait loci mapping, and marker-assisted selection in related breeding programs.

Development and characterization of 13 polymorphic microsatellite markers for the Chinese surf clam (Mactra chinensis) through Illumina paired-end sequencing

Chinese surf clam (Mactra chinensis) is a commercially important bivalve in China. The high-throughout transcriptome sequencing of M. chinensis provided a valuable resource for mining large number of microsatellite markers. In this study, we developed and characterized 13 novel polymorphic microsatellite markers for M. chinensis. The number of alleles varied from 4 to 7, with an average of 5.2 per locus. The observed and expected heterozygosity ranged from 0.1667 to 0.7368 and 0.6434 to 0.8056, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These novel informative microsatellite markers will be useful for further genetic diversity analysis, linkage mapping, evolutionary studies and marker-assisted breeding.

Genome-wide polymorphisms and development of a microarray platform to detect genetic variations in Plasmodium yoelii

The rodent malaria parasite Plasmodium yoelii is an important model for studying malaria immunity and pathogenesis. One approach for studying malaria disease phenotypes is genetic mapping, which requires typing a large number of genetic markers from multiple parasite strains and/or progeny from genetic crosses. Hundreds of microsatellite (MS) markers have been developed to genotype the P. yoelii genome; however, typing a large number of MS markers can be labor intensive, time consuming, and expensive. Thus, development of high-throughput genotyping tools such as DNA microarrays that enable rapid and accurate large-scale genotyping of the malaria parasite will be highly desirable. In this study, we sequenced the genomes of two P. yoelii strains (33X and N67) and obtained a large number of single nucleotide polymorphisms (SNPs). Based on the SNPs obtained, we designed sets of oligonucleotide probes to develop a microarray that could interrogate ∼11,000 SNPs across the 14 chromosomes of the parasite in a single hybridization. Results from hybridizations of DNA samples of five P. yoelii strains or cloned lines (17XNL, YM, 33X, N67 and N67C) and two progeny from a genetic cross (N67×17XNL) to the microarray showed that the array had a high call rate (∼97%) and accuracy (99.9%) in calling SNPs, providing a simple and reliable tool for typing the P. yoelii genome. Our data show that the P. yoelii genome is highly polymorphic, although isogenic pairs of parasites were also detected. Additionally, our results indicate that the 33X parasite is a progeny of 17XNL (or YM) and an unknown parasite. The highly accurate and reliable microarray developed in this study will greatly facilitate our ability to study the genetic basis of important traits and the disease it causes.

Microsatellite marker analysis reveals the events of the introduction and spread of cultivated mulberry in the Indian subcontinent

The secret art of silk culture along with mulberry seeds is presumed to have spread from China to other parts of the world including India through the famous Silk Road. In this study, we investigated a set of 36 important mulberry genotypes (designated as ‘breeders’ collection’) of historical importance that have been frequently used in Indian crop improvement programmes over the last five decades. This study is the first to employ a large number of microsatellite markers (140 screened; 70 used for profiling) to elucidate the diversity, structure and breeding history of mulberry. The cluster and STRUCTURE analysis corroborated with the known genetic relationships and origin. The groupings by STRUCTURE (k= 4) confirm parallel breeding efforts undertaken in the eastern, southern and northern regions of the country. The cultivar ‘Mysore Local’ shares a common parent with ‘Berhampore Local’, and this supports the historical records of its introduction from eastern India to the Kingdom of Mysore by the erstwhile ruler Tippu Sultan. The popular variety ‘Kanva-2’ shared a common male parent with the Japanese variety ‘Kousen’, supporting the contribution of exotic progenitors in Indian cultivars. The findings of this study will be useful in formulating new strategies for mulberry improvement and reveals the historical events of the introduction and spread of cultivated mulberry in the Indian subcontinent.

Development of genomic simple sequence repeat markers for linseed using next-generation sequencing technology

Linseed (Linum usitatissimum L.) is regarded as a cash crop of tomorrow because of the presence of nutraceutically important α-linolenic acid (ALA) and lignan. However, only limited breeding progress has been made in this crop, mainly due to the lack of sufficient genetic and genomic resources. Among these, simple sequence repeats (SSR) are useful DNA markers for diversity analysis, genetic mapping and tagging traits because of their co-dominant and highly polymorphic nature. In order to develop SSR markers for linseed, we used three microsatellite isolation methods, viz., PCR Isolation of Microsatellite Arrays (PIMA), 5′-anchored PCR method, and Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). The amplified products from these methods were pooled and sequenced using the 454 GS-FLX platform. A total of 36,332 reads were obtained, which assembled into 2,183 contigs and 2,509 singlets. The contigs and the singlets contained 1,842 microsatellite motifs, with dinucleotide motifs as the most abundant repeat type (54%) followed by trinucleotide motifs (44%). Based on this, 290 SSR markers were designed, 52 of which were evaluated using a panel of 27 diverse linseed genotypes. Among the three enrichment methods, the 5′-anchored PCR method was most efficient for isolation of microsatellites, while FIASCO was most efficient for developing SSR markers. We show the utility of next-generation sequencing technology for efficiently discovering a large number of microsatellite markers in non-model plants.

Negative heterozygosity-fitness correlations observed with microsatellites located in functional areas of the genome

Genome-wide heterozygosity inferred from neutral markers such as microsatellites is often expected to (i) reflect individual inbreeding and (ii) covary positively with fitness, generating positive heterozygosity-fitness correlations (HFCs). The often forgotten other end of the inbreeding-outbreeding continuum is outbreeding depression: past a certain degree of heterozygosity, heterozygotes tend to have lower fitness than homozygotes. Outbreeding depression arises from the breakup of co-adapted gene complexes and/or the introgression of nonlocally adapted genes. Provided that a correlation in heterozygosity exists across loci, outbreeding depression will be reflected in negative HFCs. In this issue, Olano-Marin et al. (2011a) describe negative heterozygosity-fitness correlations (HFCs) in blue tits Cyanistes caeruleus (Fig. 1), whereby heterozygosity has a significant, negative effect on female hatching success and recruitment. This study, together with a similar study by the same authors published in Evolution (Olano-Marin et al. 2011b), forms an original contribution in two respects. First, in the same population, positive and negative HFCs were recorded, revealing both inbreeding and outbreeding depression depending on the trait studied (whereby both processes were reliant on unknown, and possibly different, sets of coding loci). Second, a large number of microsatellite markers were split into two functional groups: microsatellite markers were either designed using zebra finch expressed sequence tags (ESTs) or derived using traditional cloning methods and presumed to be neutral. Contrasting large classes of loci and their varying levels of polymorphism, rather than looking for one locus that would stand out among tens of randomly selected markers, pave the way for a more elegant and powerful approach to explore how HFCs vary across traits and among regions of the genome. [Figure: see text].

A comparative genomic map for Caulanthus amplexicaulis and related species (Brassicaceae)

Adaptation to environment is the cornerstone of ecological genetics. The subject of this study is a wild relative of the sequenced and annotated model plant species, Arabidopsis thaliana. Caulanthus amplexicaulis var. barbarae lives on serpentine soils, known for high concentrations of heavy metals and low concentrations of essential plant macronutrients, and provides a compelling example of an organism's adaptation to environment. We constructed an F(2) linkage map, using a cross to the nonserpentine sister taxon, C.amplexicaulis var. amplexicaulis. C.amplexicaulis is a member of a highly diverse set of taxa (within the tribe Thelypodieae), described here as the 'Streptanthoid Complex' that are adapted to a broad range of environments, yet share a common n = 14 chromosome number and likely arose by a recent radiation. The linkage map consists of 97 polymorphic microsatellite markers, and 40 exon-primed intron-crossing markers based on A.thaliana exon sequences and Brassica ESTs. The map covers 14 linkage groups and has a total length of 1513 cM. Both the patterns of marker segregation and the comparative map indicate that C.amplexicaulis is a diploid organism with a compact genome. All exon-primed intron-crossing markers, and an unexpectedly large number of microsatellite markers (83%), had significant similarity to the A.thaliana genome, facilitating the development of a comparative genome map. As a proof of principle, we used the comparative map to identify candidate genes underlying differences in sepal colour between the two parent taxa. We demonstrate that the genomic tools developed here will be portable throughout the Streptanthoid Complex.

Transferability of microsatellite markers among economically and ecologically important galliform birds

We used the partially sequenced genomes of the turkey and chicken to find a large number of microsatellite markers. We then characterized 10 polymorphic microsatellite markers developed by cross-species amplification from economically and ecologically important birds to various European sub-species of the grey partridge. Even though we used cross-species amplification, a high degree of polymorphism was conserved in all microsatellite markers. Cross-species amplification from birds of economic and ecological interest, such as chicken and turkey, could be an attractive approach to develop microsatellite markers and to use these to manage wild and captive populations of other galliforms, such as the grey partridge.

Methods comparison for microsatellite marker development: Different isolation methods, different yield efficiency

Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology, quantitative genetics and genomics. Therefore, it is extremely necessary to select several versatile, low-cost, efficient and time- and labor-saving methods to develop a large panel of microsatellite markers. In this study, we used Zhikong scallop (Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency, while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time- and cost-saving, it is difficult to obtain a large number of microsatellite markers, mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study, we recommend two methods, microsatellite-enriched library construction method and FIASCO-colony hybridization method, for large-scale microsatellite marker development. Both methods were derived from the microsatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.

Genic non-coding microsatellites in the rice genome: characterization, marker design and use in assessing genetic and evolutionary relationships among domesticated groups

BackgroundCompletely sequenced plant genomes provide scope for designing a large number of microsatellite markers, which are useful in various aspects of crop breeding and genetic analysis. With the objective of developing genic but non-coding microsatellite (GNMS) markers for the rice (Oryza sativa L.) genome, we characterized the frequency and relative distribution of microsatellite repeat-motifs in 18,935 predicted protein coding genes including 14,308 putative promoter sequences.ResultsWe identified 19,555 perfect GNMS repeats with densities ranging from 306.7/Mb in chromosome 1 to 450/Mb in chromosome 12 with an average of 357.5 GNMS per Mb. The average microsatellite density was maximum in the 5' untranslated regions (UTRs) followed by those in introns, promoters, 3'UTRs and minimum in the coding sequences (CDS). Primers were designed for 17,966 (92%) GNMS repeats, including 4,288 (94%) hypervariable class I types, which were bin-mapped on the rice genome. The GNMS markers were most polymorphic in the intronic region (73.3%) followed by markers in the promoter region (53.3%) and least in the CDS (26.6%). The robust polymerase chain reaction (PCR) amplification efficiency and high polymorphic potential of GNMS markers over genic coding and random genomic microsatellite markers suggest their immediate use in efficient genotyping applications in rice. A set of these markers could assess genetic diversity and establish phylogenetic relationships among domesticated rice cultivar groups. We also demonstrated the usefulness of orthologous and paralogous conserved non-coding microsatellite (CNMS) markers, identified in the putative rice promoter sequences, for comparative physical mapping and understanding of evolutionary and gene regulatory complexities among rice and other members of the grass family. The divergence between long-grained aromatics and subspecies japonica was estimated to be more recent (0.004 Mya) compared to short-grained aromatics from japonica (0.006 Mya) and long-grained aromatics from subspecies indica (0.014 Mya).ConclusionOur analyses showed that GNMS markers with their high polymorphic potential would be preferred candidate functional markers in various marker-based applications in rice genetics, genomics and breeding. The CNMS markers provided encouraging implications for their use in comparative genome mapping and understanding of evolutionary complexities in rice and other members of grass family.

A whole genome association study in Finnish multiple sclerosis patients with 3669 markers

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with complex genetic background. In the present study, based in the Finnish population, we typed a large number of microsatellite markers in separately pooled DNA samples from 195 MS patients and 205 controls. A total of 108 markers showed evidence of association. Five genomic regions containing two or more of these markers within a 1-Mb interval were identified, 1q43, 2p16, 4p15, 4q34 and 6p21 (the MHC region). Substantial overlap with previously published linkage genome screens is also seen.

Microsatellites obtained using strand extension: an enrichment protocol.

A new method is described to enrich genomic libraries for clones containing microsatellite repeats. The method involves selection on completed M13 genomic libraries rather than on genomic DNA before library construction. It uses two reactions, in which microsatellite oligonucleotides prime strand extension. The first reaction uses a biotinylated primer allowing vectors with microsatellite-containing inserts to be selected with streptavidin-coated magnetic beads. This reaction may be dependent on the strand displacement activity of the Klenow fragment of DNA Polymerase I. The second strand extension reaction is included to improve the relative transformation efficiency of microsatellite-containing clones. In control experiments starting with 0.7% microsatellite-containing clones, enrichment averaged 99.5%. The method was tested empirically on antechinus and abalone genomic libraries in which enrichment for (CA)n microsatellites was efficient enough that clones could be sequenced without further screening. This protocol is technically straightforward and permits the isolation of a large number of microsatellite markers in less time than is required to execute traditional protocols involving rounds of filter hybridization.