Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

Abundance, polymorphism and genetic mapping of microsatellites in rice

Dinucleotide microsatellites have been characterized and used as genetic markers in rice. Screening of a rice genomic library with poly(dG-dA)·(dC-dT) and poly(dG-dT)·(dC-dA) probes indicated that (GA)_n repeats occurred, on average, once every 225 kb and (GT)_n repeats once every 480 kb. DNA sequencing of ten randomly selected microsatellites indicated that the numbers of repeats ranged from 12 to 34 and that the patterns of microsatellites in rice were similar to those of humans and other mammals. Primers to these microsatellite loci as well as to four published microsatellite-containing sequences have been designed and degrees of polymorphism has been examined with 20 rice accessions. Multiple alleles, ranging from 5 to 11, have been observed at all the microsatellite loci in 20 rice accessions. Alleles specific to two cultivated subspecies, indica and japonica, were found in some microsatellite loci. Heterozygosity values of all the microsatellite markers were significantly higher than those of RFLP markers, based upon a parallel comparison. Ten microsatellite loci have been genetically mapped to four rice chromosomes. The genomic distribution of microsatellites appears to be random in rice.     

The human protein translin specifically binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n: a study of the binding parameters

We have previously identified in human fibroblasts a multisubunit protein (designated PGB) that specifically bound single-stranded G-rich microsatellite DNA sequences. PGB was later found to be identical, or closely related to translin, an octameric protein that bound single-stranded DNA consisting of sequences flanking chromosomal translocations. Here, we report that recombinant translin binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n, with higher affinities (Kdis approximately = 2 nM and Kdis approximately = 12.5 nM, respectively, in 100 mM NaCl and 25 degrees C) than the affinity with which it binds a prototypical sequence flanking translocation sites (Kdis approximately = 23 nM). Translin also binds d(GT)n and d(TTAGGG)n overhangs linked to double-stranded DNA with equilibrium constants in the nanomolar range. Formation of DNA quadruplexes by the d(TTAGGG)n repeats inhibits their binding to translin. A further study of the binding parameters revealed that the minimal length of d(GT)n and d(TTAGGG)n oligonucleotides that a translin octamer can bind is 11 nucleotides, but that such oligonucleotides containing up to 30 nucleotides can bind only a single translin octamer. However, the oligonucleotides d(GT)27 and d(TTAGGG)9 bind two octamers with negative cooperativity. Translin does not detectably bind single-stranded d(GT)n sequences embedded within double-stranded DNA. Based on our data, we propose that translin might be involved in the control of recombination at d(GT)n.d(AC)n microsatellites and in telomere maintenance.     

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     

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides (CT)10 and (GT)10 served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.     

Low abundance of microsatellite repeats in the genome of the brown-headed cowbird (Molothrus ater).

A cosmid library made from brown-headed cowbird (Molothrus ater) DNA was examined for representation of 17 distinct microsatellite motifs including all possible mono-, di-, and trinucleotide microsatellites, and the tetranucleotide repeat (GATA)n. The overall density of microsatellites within cowbird DNA was found to be one repeat per 89 kb and the frequency of the most abundant motif, (AGC)n, was once every 382 kb. The abundance of microsatellites within the cowbird genome is estimated to be reduced approximately 15-fold compared to humans. The reduced frequency of microsatellites seen in this study is consistent with previous observations indicating reduced numbers of microsatellites and other interspersed repeats in avian DNA. In addition to providing new information concerning the abundance of microsatellites within an avian genome, these results provide useful insights for selecting cloning strategies that might be used in the development of locus-specific microsatellite markers for avian studies.     

Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)n.(dG-dT)n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)n.(dG-dT)n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)n.(dG-dT)n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)n.(dG-dT)n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)n.(dG-dT)n polymorphisms regardless of the repeat sequence category.     

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     

Polymorphic microsatellites in a mangrove species,Rhizophora mangle L. (Rhizophoraceae)

Twenty‐six microsatellite loci were isolated and characterized from the mangrove species Rhizophora mangle using (GT)_n and (CT)_n repeats. Eighty‐four per cent of the clones contained microsatellite sequences; the most common dinucleotides were the (GA/CT) and (CA/GT) repeats. Ten primers were selected to investigate the polymorphism among individuals of R. mangle from two natural populations of the Colombian Pacific Coast. The observed heterozygosity per locus varied from 0.20 to 0.80, the power of discrimination was 0.32–0.84 and the power of exclusion was 0.03–0.75. This set of microsatellites offers an efficient tool for population genetics studies on this species.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

Development of simple sequence repeat markers in rye (Secale cereale L.).

Simple sequence repeats (SSRs), also referred to as microsatellites, represent a PCR-based marker system that has been described in mammalian and plant genomes in recent years. In self-pollinating crop plants they have been shown to be superior to other DNA markers with respect to their level of polymorphism. The technical advantages compared with RFLP markers should also facilitate marker analysis in outcrossing crops like rye. In order to determine the usefulness of SSR markers in rye genetics and breeding, several genomic libraries were screened for (CT/GA)n and (GT/CA)n dinucleotide repeats. It was estimated that these motifs occur at a frequency of one per 268-519 kb. Seventy four out of 182 positive clones were sequenced, and the majority (56.8%) revealed perfect repeats, predominantly of the type (GT/CA)n (61.9%). Fifty seven primer pairs were designed and 27 (47.4%) resulted in specific SSR markers, of which 20 were genetically mapped or assigned to chromosomes or chromosome arms, respectively. The level of polymorphism of four SSR and three RFLP markers was assessed in two open-pollinated rye cultivars. On average, the SSR markers showed larger values of expected heterozygosity (0.62 vs. 0.43) and allele number (5.9 vs. 3.4) than RFLP markers in both cultivars.     

Microsatellite repeats are not randomly distributed within Norway spruce (Picea abies K.) expressed sequences.

A Norway spruce (Picea abies K.) cDNA library obtained from vegetative bud tissue was screened for the presence of (AG)n and (AC)n microsatellite repeats. Ten (AG)n and six (AC)n microsatellites were found, with an average length of 25.5 repeat units. Most of the microsatellites are simple perfect repeats. The microsatellite distribution within the clones is clearly non-random, with different classes of repeats lying in different positions relative to the coding region and in a highly conserved orientation. An estimate of the frequency of dinucleotide microsatellites in expressed regions was obtained, showing that SSRs (simple sequence repeats) are found in genes about 20 times less frequently than in random genomic clones, with (AG)n repeats more frequent than (AC)n repeats. Potential applications of these sequences as expressed region-based molecular markers are shown by developing six SSR markers for the detection of natural variation in Norway spruce populations and testing two of them for the identification of illegitimate progenies from a mapping population.     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

Microsatellites in Malus X domestica (apple): abundance, polymorphism and cultivar identification

Screening of an apple genomic library with (GA)15 and (GT)15 probes demonstrated that these repeats are abundant, occurring about every 120 and 190 kb, respectively. Microsatellites isolated from a small insert library enriched for (GA) repeats contained numbers of repeats ranging from 7 to 39. Primers to these microsatellite loci were able to direct the amplification of the repeats in 21 different cultivars. The majority of markers were highly polymorphic, diploid, and showed simple Mendelian inheritance, although about 25% of markers generated complex banding patterns consistent with the amplification of more than one locus. As few as three microsatellite markers were sufficient to differentiate between all 21 cultivars. Received: 20 February 1996 / Accepted: 24 May 1996     

Interspecific Utility of Microsatellites in Fish: A Case Study of (CT)n and (GT)n Markers in the Shanny Lipophrys pholis (Pisces: Blenniidae) and Their Use in Other Blennioidei

We report the development of new microsatellite markers that can be used for population analyses in the shanny Lipophrys pholis. The procedure involved the construction of a microsatellite-enriched genomic bank. Five (GT)_n and (CT)_n microsatellites have been characterized, four of which are polymorphic. The analysis of one population allowed us to verify their usefulness as markers in population studies. Moreover, interspecific amplifications have been performed using primers defined in other species to amplify Lipophrys pholis, or using the primers defined in Lipophrys pholis to amplify other species. We use these results to discuss the hypothesis that microsatellites are highly conserved in fish. Received May 13, 1999; accepted October 28, 1999.     

Isolation and characterization of polymorphic microsatellites in the genome of Yak (Bos grunniens)

The researches on yak genetics and breeding were extremely restricted due to lacking of reliable DNA molecular markers. The microsatellites with repeat motif (AC)_n/(GT)_n in yak genome were enriched by Dynal magnetic beads and the gene libraries containing (AC)_n/(GT)_n were constructed. Among the 92 identified and sequenced positive clones, 40 contained perfect repeats (43.48 %), 41 contained imperfect repeats (44.57 %) and 11 contained compound repeats (11.96 %). As compared with the percentage of perfect repeats, no significant increases of imperfect repeats were observed in yak genome, which indicated that the level of adaptive evolution of the ability to repair damaged genomic DNA for yaks were high enough to endure the natural pressure of nucleotide substitution resulted from ultraviolet irradiation in high-altitude areas. Totally 19 polymorphic microsatellite loci were screened and genotyped on the basis of electropherograms on an ABI 310 Genetic Analyzer. All the loci exhibited moderate to high-level polymorphisms in a test population of Bos grunniens and the polymorphic information content ranged from 0.299 to 0.861 (mean 0.678). The newly isolated (AC)_n/(GT)_n repeats from yak genome will display their potential values in examining intra-population genetic structure and inter-population relationships, and also in investigating molecular markers for production and adaptive traits of individual/population.     

Survey of human and rat microsatellites

Length variations in simple sequence tandem repeats (microsatellite DNA polymorphisms) are finding increasing usage in mammalian genetics. Although every variety of short tandem repeat that has been tested has been shown to exhibit length polymorphisms, little information on the relative abundance of the different repeat motifs has been collected. In this report, summaries of GenBank searches for all possible human and rat microsatellites ranging from mononucleotide to tetranucleotide repeats are presented. In humans, the five most abundant microsatellites with total lengths for the runs of repeats of greater than or equal to 20 nucleotides contained repeat sequences of A, AC, AAAN, AAN, and AG, in order of decreasing abundance, where N is C, G, or T. These five groups comprised about 76% of all microsatellites. Many other human simple sequence repeats were found at low frequency. In the 745 kb of human genomic DNA surveyed, one microsatellite of greater than or equal to 20 nucleotides in length was found, on average, every 6 kb. Only 12% of the human microsatellites had total lengths greater than or equal to 40 nucleotides. Roughly 80% of the A, AAN, and AAAN microsatellites and 50% of the AT microsatellites, but few of the other human microsatellites, were found to be associated with interspersed, repetitive Alu elements. In rats, the five most abundant microsatellites contained AC, AG, A, AAAN, and AAGG sequences, respectively. Rat microsatellites were generally longer than human microsatellites, with 43% of the rat sequences greater than or equal to 40 nucleotides.     

Conformational changes induced in the human protein translin and in the single-stranded oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5) upon binding of these oligodeoxynucleotides by translin

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.