Tetranucleotide Repeat Motifs Research Articles

Development of Microsatellite Markers for the Korean Mussel, Mytilus coruscus (Mytilidae) Using Next-Generation Sequencing

Mytilus coruscus (family Mytilidae) is one of the most important marine shellfish species in Korea. During the past few decades, this species has become endangered due to the loss of habitats and overfishing. Despite this species’ importance, information on its genetic background is scarce. In this study, we developed microsatellite markers for M. coruscus using next-generation sequencing. A total of 263,900 raw reads were obtained from a quarter-plate run on the 454 GS-FLX titanium platform, and 176,327 unique sequences were generated with an average length of 381 bp; 2569 (1.45%) sequences contained a minimum of five di- to tetra-nucleotide repeat motifs. Of the 51 loci screened, 46 were amplified successfully, and 22 were polymorphic among 30 individuals, with seven of trinucleotide repeats and three of tetranucleotide repeats. All loci exhibited high genetic variability, with an average of 17.32 alleles per locus, and the mean observed and expected heterozygosities were 0.67 and 0.90, respectively. In addition, cross-amplification was tested for all 22 loci in another congener species, M. galloprovincialis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 22 newly developed microsatellites will be useful in future conservation genetic studies of this species.

The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome

Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene. The expansions are extremely unstable and variable, ranging from 75–11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3′-end of an AluSx element, flanked by target site duplications (5′-ACTRCCAR-3′or 5′-ACTRCCARTTA-3′), followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG) repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A) tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s) by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders.

Data mining of ESTs to develop dbEST-SSRs for use in a polymorphism study of cauliflower (Brassica oleracea var. botrytis)

SummaryExpressed sequence tags (ESTs) provide a valuable resource for developing simple sequence repeat (SSR; microsatellite) markers. In this study, 3,364 EST sequences belonging to four varieties of Brassica oleracea (botrytis cauliflower), capitata (cabbage), italica (broccoli), and gemmifera (brussels sprout) and to Arabidopsis thaliana, derived from the NCBI website (http://www.ncbi.nlm.nih.gov/nucest), were screened for SSR motifs using two search programmes, MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html) and SSRIT (http://www.gramene.org/db/markers/ssrtool). A total of 76 ESTs were found to contain tri-nucleotide or tetra-nucleotide repeat motifs, with AAC and ACAT being in greatest abundance. Using PRIMER3 software (www.frodo.wi/mit.edu/primer3), 62 primer pairs were designed for further use as potential genic markers. Of this set, 16 primer pairs were custom synthesised and used in polymorphism studies on cauliflower. Annotation analysis using BLASTX revealed that a putative function could be assigned to 15 of the 16 EST-SSRs selected for primer design. Thirteen of these 16 markers gave scorable bands, 11 of which revealed moderate-to-high polymorphism information content (PIC) values across 20 genotypes of cauliflower. Eighteen genomic SSR primers were also used in this study, out of which 16 primer pairs amplified cauliflower DNA, eight primers being polymorphic. Cluster analysis based on dendrograms formulated using the UPGMA algorithm revealed the genetic distances between genotypes, which so far have been based only on morphological traits. The new set of EST–SSR markers described in this study should be useful for further studies on genetic diversity and genome analysis in this major crop species.

Microsatellite markers for genetic studies in the weasel (Mustela nivalis)

This study reports the first set of microsatellite markers for the weasel (Mustela nivalis). We chose to isolate loci with tetranucleotide repeat motifs because they can be scored less ambiguously than the more commonly used dinucleotide loci. All 11 loci showed considerable variation within a population sample of 28 individuals from Portugal, with number of alleles ranging from four to nine per locus and observed and expected heterozygosities ranging from 0.21 to 0.86 and from 0.40 to 0.84, respectively. No linkage disequilibrium was detected between pairs of loci, and only one locus (Mn 1.30) deviated from Hardy–Weinberg equilibrium expectations in the analyzed population sample. Among the 11 loci, Mn 1.30 was the only one for which all known males were homozygous. Analysis of an additional population sample of 23 individuals (14 males and 9 females) from Denmark revealed that all males, but only four females, were homozygous for Mn 1.30, supporting the idea that the locus is X-linked. These novel polymorphic microsatellite markers should be useful in studies of population genetics and molecular ecology of the weasel.

Tetranucleotide microsatellites contribute to a highly discriminating parentage test panel in pig

Here, we report genotyping conditions for 434 new polymorphic pig microsatellite markers containing trinucleotide and tetranucleotide repeat motifs in pig. Microsatellite sequences were detected in silico from bacterial artificial chromosome (BAC) clone end sequences and mapped to the pig genome. A set of 22 microsatellites is described, which can be separated in a simultaneous electrophoresis by multiplexing across a large size range, in combination with 4-colour labelling. Marker information content and false pedigree exclusion probabilities are documented in five purebred populations, allowing assessment of this panel in pig parentage testing applications. Combined exclusion probabilities >99.7% were achieved in all pedigree test cases.

EST‐derived microsatellites as a rich source of molecular markers for oats

AbstractPolymerase chain reaction‐based microsatellite markers are valuable tools for molecular breeding because of their co‐dominant inheritance and their applicability for high‐throughput analysis. Being still very limited for oats, their number has to be increased significantly to cover the entire genome. For these purposes, a set of 7031 recently published expressed sequence tags (ESTs) was screened for microsatellites with dinucleotide, trinucleotide and tetranucleotide repeat motifs. Subsequent in silico analysis resulted in the development of 216 primer pairs for Avena EST‐derived microsatellite loci (AME). Using a sample set of 12 oat lines, 107 of 195 functional primers could be assayed as polymorphic. The marker variability averaged out at three alleles per locus and a polymorphic information content (PIC) value of 0.42. This variability documents their suitability for molecular oat‐breeding purposes. Finally, 51 of the AME loci could be placed within the known reference map of ‘Kanota’ × ‘Ogle’ that previously contained only 12 microsatellite loci. Thus, a remarkable enhancing of the number of mapped oat microsatellite loci could be achieved.

Development and characterization of 248 novel microsatellite markers in turbot (Scophthalmus maximus)

The turbot is a flatfish species of great relevance to marine aquaculture in Europe. Only a limited number of microsatellites have been isolated to date in this species. To increase the number of potentially useful mapping markers, we screened simple sequence repeat (SSR)--enriched genomic libraries obtained from several di-, tri-, and tetranucleotide tandem repeat motifs. A total of 248 new polymorphic microsatellites were successfully optimized. The efficiency of the protocol applied (6.4%) was higher than that in other studies of fish that used the same method. Dinucleotide and perfect microsatellites were predominant in this species; the (AC)n motif was the most frequent class of repeat. Polymorphism and structural properties at these loci, together with 30 variable loci previously reported in turbot, were evaluated in 6 wild individuals. The number of alleles per locus ranged from 2 to 10, with an average of 4.046. The microsatellite markers characterized in this study will contribute to the development of the turbot genetic map, which can be used for quantitative trait locus (QTL) identification, marker-assisted selection programs, and other applications to improve its culture.

Microsatellite Loci from Lanyu Scops Owl (Otus elegans botelensis) and their Cross-Species Application in Four Species of Strigidae

We developed six new microsatellite markers containing tetranucleotide repeat motifs (GATA/CTAT) for Lanyu scops owl (Otus elegans botelensis) from an enriched partial library. All these loci are polymorphic and conform to Hardy–Weinberg equilibrium. We cross-species tested these and 12 other microsatellite primer pairs previously developed from O. elegans on four other species of owls (O. lettia, O. spilocephalus, O. scops, and Ninox scutulata). Results showed that the degree of polymorphism decreased with increasing phylogenetic distance to O. elegans. Most loci (66.7, 83.3, and 100%) were polymorphic in the three Otus owls but only five (27.8%) were polymorphic in N. scutulata. These microsatellites should be very useful genetic markers in studying the mating system, population genetics, and conservation of other little studied Old World Otus owls.

Polymorphism of LPL Locus in Japanese and Comparison of PCR Amplification Efficiency from Degraded DNA between LPL Locus and the D21S11

The short tandem repeat (STR) polymorphism of the lipoprotein lipase (LPL) locus was amplified by PCR and analyzed using denaturing polyacrylamide gel electrophoresis followed by silver staining. Among 158 DNA samples from the Japanese population, six alleles were observed. When the sequences of the allelic products were compared, each allelic segment contained 7 and 9-13 TTTA tetranucleotide repeat motifs. Genotypic distribution met Hardy-Weinberg expectations, and included heterozygosity was 48.8%. Most of the Japanese genotypes allele 10. When PCR amplification efficiency for the LPL locus from degraded DNA was compared with that for the D21S11 locus in terms of amplification size, increase in amplification size showed a considerable influence on amplification efficiency, producing inaccurate amplification, such as unbalanced amplification, or amplification of non-target PCR products. These results suggest that reduction in amplification size increases the accuracy and efficiency of PCR amplification from highly degraded DNA.

Low-copy microsatellite recovery from a conifer genome

Microsatellite development has been stymied by highly repetitive DNA in the large, highly duplicated conifer genome and by so few genomic conifer sequences in public databases. Recovery of microsatellites from the low-copy component was tested as an efficient approach to marker development. Microsatellites were isolated from Pinus taeda L. via low-copy enrichment and filter-hybridization of tri- and tetra-nucleotide repeat motifs. Efficiency at three phases of marker development was compared for low-copy and total-genome control libraries. In the first phase, enrichment for microsatellites was slightly lower in the low-copy libraries. In the second phase, redundancy was higher in the low-copy libraries. In the third phase, low-copy libraries provided more polymorphic markers than total-genome libraries. Of 418 sequenced low-copy clones, 102 were unique sequences with repeat motifs. Of these unique sequences, twice as many were useful for marker development compared to the total-genome control. Difficulty in microsatellite marker development due to highly repetitive DNA can be abated by low-copy enrichment or circumvented by selecting for specific CG-rich trinucleotide repeat motifs. Sixteen new low-copy and genomic P. taeda microsatellites were given as an example.

Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability

Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the “classic pathway” of colorectal cancer, i.e. p53 and ras, and also in the “mutator pathway” by evaluating microsatellite instability. Exons 5–9 of the p53 gene and exons 1 and 2 of the K- ras and H- ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.

Northern blot analysis of simple repetitive sequence transcription in plants

The presence of simple repetitive sequence motifs in RNA from various plant species was probed by Northern blot analysis. Hybridization of total, poly(A)(+)- and poly(A)(-)-RNA with microsatellite-complementary oligonucleotide probes revealed distinct bands with most but not all probe/species combinations, demonstrating the presence of di-, tri- and tetranucleotide repeat motifs in plant transcripts. Only trinucleotide repeat-derived hybridization signals were found to be enriched in the poly(A)(+)-fraction. The quality of Northern blot signals proved to be highly dependent on hybridization stringency. Thus, under the stringency conditions usually applied for oligonucleotide hybridization, some probes [(GT)8, (CAC)5, (TCC)5, and (CCTA)4] cross-hybridized to bands corresponding in size to 18S and/or 26S rRNA. Cross-hybridization to rRNA was significantly reduced at higher stringencies. These results stress the importance of carefully adjusting the hybridization conditions in Northern blot analysis of simple sequence transcripts.

Frequency of microsatellite sequences in rice (Oryza sativa L.).

This study was undertaken to estimate the relative frequencies of 13 microsatellite motifs in the rice genome as a basis for efficient development of a microsatellite map. Two dinucleotide, seven trinucleotide, and four tetranucleotide repeat motifs were end labelled and used as hybridization probes to screen genomic and cDNA libraries of rice, cv. IR36. Optimal washing temperatures for identification of clones containing specific microsatellite motifs were estimated based on washing temperatures near Td (dissociation temperature; Td = Tm - 7.6 degrees C). Sequencing of 20 putatively positive clones corresponding to each of 4 microsatellite motifs suggested that while Td provides a useful predictor of washing stringency for most of the repeats studied, those with a very high GC or AT content were most prone to error. The results from screening the rice genomic library suggest that there are an estimated 1360 poly(GA)n and 1230 poly(GT)n microsatellites in the rice genome, and that the relative frequency of different repeats decreased with increasing size of the motif. The most frequently observed microsatellites in the cDNA library were the same as for genomic library, but no poly(CGG)n, poly(ATC)n, or tetranucleotide motifs were observed among cDNA in this study.

Integration of 28 STSs into the physical map of human chromosome 18.

Genes on human chromosome 18 are associated with familial glucocorticoid deficiency (MC2R), pemphigus vulgaris (DSG3) and foliaceus (DSG1), familial amyloidosis (TTR), colorectal carcinoma (DCC), erythropoietic protoporphyria (FECH), follicular lymphoma (BCL2, FVT1), and congenital methemoglobinemia (CYB5). As the resolution of human genetic maps improves, linkage between other diseases and specific regions of chromosome 18 will occur. A physical map of human chromosome 18 will prove useful in identifying candidate genes that are associated with these disorders. Using various physical and genetic mapping techniques, over 35 genes and 19 expressed sequence tags (ESTs) are assigned to human chromosome 18. Most of these genes and several of the ESTs were sublocalized using a well-defined panel of somatic cell hybrids that contain different segments of human chromosome 18. Despite recent efforts, progress in mapping human chromosome 18 has lagged behind that achieved for other chromosomes. Thus, the purpose of this study was to integrate 9 new transcriptional tags [8 brain ESTs (8) and the melanocortin 4 receptor (MC4R) (3)] and 19 simple sequence repeats (SSRs) into the physical map of human chromosome 18. The SSRs were isolated by screening genomic DNA libraries constructed in M13mp18 vectors with oligonucleotide probes that detected dinucleotide d(CA)- andmore » tetranucleotide-repeat motifs. DNA sequences of clones that contained microsatellite repeats were obtained by thermal-cycle sequencing, and STSs were developed from clones that contained numerous repeats. STSs that identified highly polymorphic loci in eight unrelated CEPH parents were used for genotyping. Results of linkage analyses and estimates of heterozygosity for these markers will be reported. 9 refs., 1 fig., 1 tab.« less