Trinucleotide Microsatellite Research Articles

Microsatellite loci from the trematode Lecithochirium fusiforme, a parasite of the European conger eel

AbstractSeven polymorphic loci containing dinucleotide repeats and one trinucleotide microsatellite were developed for the hemiurid fluke Lecithochirium fusiforme, a parasite of the European conger eel Conger conger. All parasites that were collected from a single individual host (a total of 54 specimens) were genotyped. The number of alleles ranged from two to eight. The observed and expected heterozygosities ranged from 0.057 to 0.736 and from 0.091 to 0.794, respectively. Hardy–Weinberg deviations were statistically significant for two loci. These markers will be useful for study of parasite transmission patterns and population genetic structure.

Characterisation of Teladorsagia circumcincta microsatellites and their development as population genetic markers

There is a need to develop tools to study the genetics of parasitic nematodes. This is particularly urgent for those species in which anthelmintic resistance is common such as the sheep parasite Teladorsagia ( Ostertagia) circumcincta. The lack of information on the population genetics of such parasites severely limits our ability to study the genetic basis of anthelmintic resistance. This paper presents the results of three approaches used to isolate microsatellite markers from T. circumcincta and the development of a panel of markers suitable for population genetic analysis. Hybridisation screening of small insert genomic libraries and interspecies PCR amplification of Haemonchus contortus microsatellites were used to identify CA/GT microsatellites. Many of these loci were associated with a 146 bp tandem repeat, named TecRep, that is related to a repetitive element previously identified in other trichostrongylid nematode genomes but apparently absent from other nematode groups. A large proportion of the loci isolated were problematic for use as population genetic markers, predominantly due to a high frequency of null alleles or the association with the TecRep repeat. Bioinformatic screening of a T. circumcincta EST database identified both di- and tri-nucleotide microsatellite repeats and a greater proportion of these turned out to be more robust markers than those derived from genomic sequence. A panel of seven markers has been selected and characterised which are sufficiently robust and polymorphic to be valuable population genetic markers for this parasite.

Global distribution of a novel trinucleotide microsatellite polymorphism (ATA)n in intron 8 of the SLC11A1 gene and susceptibility to pulmonary tuberculosis

We identified a novel trinucleotide (ATA)n repeat polymorphism in intron 8 of SLC11AI, a candidate gene for susceptibility to tuberculosis (TB) infection. We characterized the frequency of this polymorphism in 485 individuals originating from eight globally diverse human populations and compared the distribution of (ATA)n alleles in 146 adults and in 80 cord blood samples from newborns in the Gambian population. Lastly, we tested for association of this microsatellite with pulmonary TB in 318 TB cases and 146 controls in the Gambian population. We found no significant difference in frequency or distribution of alleles in adult and cord blood samples, and we found no significant association between this marker and pulmonary TB.

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as 'genome renewal'.

Microsatellite markers derived from bay scallop Argopecten irradians expressed sequence tags

When data mining was performed on the National Center for Biotechnology Information database, a total of 2038 sequences from five different expressed sequence tag libraries were registered. Eighty sequences (3.9%) were found to contain 91 microsatellites. Clustering analysis indicated that 23 sequences of these expressed sequence tags fell into five clusters and that the remaining 57 sequences were independent. The di- and tri-nucleotide repeat motifs accounted for approximately 62.1% of the total microsatellites. The most abundant dinucleotide microsatellite was TA, followed by GA and CA, and the trinucleotide microsatellites GAT and GGT showed a high abundance. Nineteen sequences representing di-, tri-, tetra- and penta-nucleotides motifs were chosen for the design of polymerase chain reaction (PCR) primers. Of primer pairs, 16 successfully amplified scorable PCR products and 11 revealed polymorphism, with the average polymorphic information content value of 0.5082 and 3.1 alleles per locus. A transferability analysis on three other related scallop species, Chlamys farreri, Chlamys nobilis and Patinopecten yessoenssis, showed that only 1 of 16 primer pairs could amplify PCR products with the expected size in Chlamys nobilis.

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract A novel set of five polymorphic di- or trinucleotide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H. assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.

Ten polymorphic microsatellite markers developed in the masson pine moth Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae)

AbstractA set of 10 polymorphic di‐ and trinucleotide microsatellite loci were developed for the forestry pest insect, masson pine moth, Dendrolimus punctatus Walker. The expected heterozygosity at these loci ranges from 0.285 to 0.859, and the observed allele numbers from five to 19. Cross species amplification of these loci in four other congeneric pine moth species indicates variable levels of loci conservation and thus cross‐applicability. Therefore, the microsatellite loci reported here should be useful for population genetic and other related studies in the masson pine moth and other closely related species.

Microsatellite Markers in Hazelnut: Isolation, Characterization, and Cross-species Amplification

Three microsatellite-enriched libraries of the european hazelnut (Corylus avellana L.) were constructed: library A for CA repeats, library B for GA repeats, and library C for GAA repeats. Twenty-five primer pairs amplified easy-to-score single loci and were used to investigate polymorphism among 20 C. avellana genotypes and to evaluate cross-species amplification in seven Corylus L. species. Microsatellite alleles were estimated by fluorescent capillary electrophoresis fragment sizing. The number of alleles per locus ranged from 2 to 12 (average = 7.16) in C. avellana and from 5 to 22 overall (average = 13.32). With the exception of CAC-B110, di-nucleotide SSRs were characterized by a relatively large number of alleles per locus (≥5), high average observed and expected heterozygosity (Ho and He > 0.6), and a high mean polymorphic information content (PIC ≥ 0.6) in C. avellana. In contrast, tri-nucleotide microsatellites were more homozygous (Ho = 0.4 on average) and less informative than di-nucleotide simple sequence repeats (SSRs) as indicated by a lower mean number of alleles per locus (4.5), He (0.59), and PIC (0.54). Cross-species amplification in Corylus was demonstrated. These microsatellite markers were highly heterozygous and polymorphic and differentiated among genotypes of C. avellana irrespective of geographical origin. They will aid in fingerprinting genotypes of the european hazelnut and other Corylus species, genome mapping, and genetic diversity assessments.

In-depth sequence analysis of the tomato chromosome 12 centromeric region: identification of a large CAA block and characterization of pericentromere retrotranposons

We sequenced a continuous 326-kb DNA stretch of a microscopically defined centromeric region of tomato chromosome 12. A total of 84% of the sequence (270 kb) was composed of a nested complex of repeat sequences including 27 retrotransposons, two transposable elements, three MITEs, two terminal repeat retrotransposons in miniature (TRIMs), ten unclassified repeats and three chloroplast DNA insertions. The retrotransposons were grouped into three families of Ty3-Gypsy type long terminal repeat (LTR) retrotransposons (PCRT1-PCRT3) and one LINE-like retrotransposon (PCRT4). High-resolution fluorescence in situ hybridization analyses on pachytene complements revealed that PCRT1a occurs on the pericentromere heterochromatin blocks. PCRT1 was the prevalent retrotransposon family occupying more than 60% of the 326-kb sequence with 19 members grouped into eight subfamilies (PCRT1a-PCRT1h) based on LTR sequence. The PCRT1a subfamily is a rapidly amplified element occupying tens of megabases. The other PCRT1 subfamilies (PCRT1b-PCRT1h) were highly degenerated and interrupted by insertions of other elements. The PCRT1 family shows identity with a previously identified tomato-specific repeat TGR2 and a CENP-B like sequence. A second previously described genomic repeat, TGR3, was identified as a part of the LTR sequence of an Athila-like PCRT2 element of which four copies were found in the 326-kb stretch. A large block of trinucleotide microsatellite (CAA)n occupies the centromere and large portions of the flanking pericentromere heterochromatin blocks of chromosome 12 and most of the other chromosomes. Five putative genes in the remaining 14% of the centromere region were identified, of which one is similar to a transcription regulator (ToCPL1) and a candidate jointless-2 gene.

Trinucleotide microsatellite loci in the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae)

AbstractWe describe six polymorphic, trinucleotide microsatellite loci that were isolated from the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae), a model system for examining the mechanisms behind sperm competition in species with internal fertilization. These microsatellites yielded between 3 and 11 alleles per locus in a sample of 20–48 dung flies collected from several sites in Cheshire, UK. Observed and expected heterozygosities varied between 0.195 and 0.900 and 0.335–0.842, respectively. These markers should allow us to investigate postcopulatory sexual selection processes in this fly much more fully than has been possible in the past.

Isolation and characterization of microsatellite markers in Phytophthora ramorum, the causal agent of sudden oak death

AbstractWe describe specific primers and conditions to amplify two dinucleotide and five trinucleotide microsatellite DNA loci isolated from the oomycete Phytophthora ramorum, the causal agent of sudden oak death. The primer sets were tested on 14–30 isolates from North America and Europe. Seven of 14 loci differentiated between A1 and A2 mating types. All seven loci successfully amplified DNA isolated from infected plant tissue. Four loci may be useful for the diagnosis of P. ramorum because they do not amplify closely related Phytophthora species.

Evolutionary history of Caenorhabditis elegans inferred from microsatellites: evidence for spatial and temporal genetic differentiation and the occurrence of outbreeding.

Although diverse biological disciplines employ the nematode Caenorhabditis elegans as a highly efficient laboratory model system, little is known about its natural history. We investigated its evolutionary past using 10 polymorphic trinucleotide and tetranucleotide microsatellites, derived from across the whole genome. These microsatellites were analyzed from the 35 previously available natural isolates from different parts of the world and also 23 new strains isolated from northwest Germany. Our results highlight that C. elegans lineages differentiate genetically with respect to geographic distance and, to a lesser extent, differences in the time of strain isolation. The latter indicates some turnover of strain genotypes at specific locations. Our data also demonstrate the coexistence of highly diverse genotypes in the population from northwest Germany, which is best explained by recent migration events. Furthermore, selfing is confirmed as the primary mode of reproduction for this hermaphroditic nematode in nature. Importantly, we also find evidence for the occurrence of occasional outbreeding. Taken together, these results support the previous notion that C. elegans is a colonizer, whereby selfing may permit rapid dispersal within new habitats even in the absence of potential mates, whereas occasional outcrossing may serve to compensate for the disadvantages of inbreeding. Such information about the natural history of C. elegans should be of great value for an in-depth understanding of the complexity of this organism, including its multifaceted developmental, neurological, or molecular genetic pathways.

Eight polymorphic microsatellite loci for the critically endangered crested ibis, Nipponia nippon (Ciconiiformes: Threskiornithidae)

AbstractThe Chinese crested ibis, Nipponia nippon, is a critically endangered bird. The current population of this species has developed from four wild individuals rediscovered in 1981. Given its conservation status, there is considerable interest in assessing the genetic diversity and individual relatedness in this species. For this purpose, a set of eight polymorphic di‐ or trinucleotide microsatellite loci was developed for the crested ibis. The expected heterozygosity at these loci ranges from 0.01 to 0.50, with less than four alleles being observed at individual loci, a reflection of the serious population bottleneck experienced by this species.

Inverted Gcg/CGC trinucleotide microsatellites in the 5'-region of Mus IDS mRNA: recurrent induction of aberrant reverse transcripts.

An investigation was initiated to explore previously published results indicating that approximately 80 bp of the 5'-end of the iduronate sulfatase (IDS) cDNA sequence (Accession No L07291) are 100% homologous with the 3'-UTR of isoform I of the sodium hydrogen exchanger (Acc. No. U51112). 5'-RACE carried out on IDS mRNA demonstrated the apparent homology to be a cloning artifact. A sequence comparison of the IDS 5'-RACE product with a mouse BAC clone covering the region, and with various IDS ESTs, suggested that the region is highly susceptible to cloning artifacts, a common one of which is template switching by reverse transcriptase. The nucleotide sequence flanking the translation start site is unusual in containing two inverted repeats composed of the complementary trinucleotide microsatellites, (GCG)9 and (CGC)6. These likely form a highly stable stem of 20-21 nt, through which reverse transcription is compromised. Such a stem could be involved in the regulation of IDS expression by directly affecting translation, message turnover, or serving as a substrate for siRNA production. Though such mRNA features are relatively rare, they may be more abundant but overlooked due to difficulties in their reverse transcription.

Ten polymorphic microsatellite DNA loci for paternity and population genetics analysis in the fen raft spider (Dolomedes plantarius)

AbstractTen polymorphic di‐ and trinucleotide microsatellite loci were developed in the fen raft spider Dolomedes plantarius from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.62 to 0.9, with the observed allele numbers varying from four to 15 in the 22 individuals tested. Average paternity exclusion probabilities ranged between 0.290 and 0.686. In combination, the 10 polymorphic loci elicit an exclusion probability of 0.999. The high level of polymorphism of these microsatellite loci makes them ideal genetic markers for paternity and population genetics analysis in this endangered species.

Typing dinucleotide repeat loci using microplate array diagonal gel electrophoresis: proof of principle.

Polymorphic dinucleotide repeat loci ('microsatellite markers') are found in varying abundance throughout the genomes of most organisms. They have been extensively used for genetic studies, but conventional techniques used for their genotyping require sophisticated equipment. Microplate array diagonal gel electrophoresis (MADGE) has previously been extended to economical high-throughput genotyping of trinucleotide and tetranucleotide microsatellite amplicons. However, the capability of this technique to resolve the alleles of dinucleotide repeat loci has not been explored previously. Here we show that a modified microsatellite-MADGE approach can provide sufficient resolution for dinucleotide repeat typing. This enables economical and convenient set up for analysis of single markers in many samples in parallel, suitable, for example, for population association studies.

Trinucleotide microsatellite loci for the peppered moth (Biston betularia)

AbstractFourteen polymorphic trinucleotide microsatellites were isolated in the peppered moth, Biston betularia, using an enrichment protocol. Moderate to high allelic diversity (three to 22 alleles per locus) was found in a sample from northern England. We are currently using these markers to study the population structure of B. betularia in northern England, where a cline for melanism still exists.

Characteristics of Microsatellites in Brassica rapa Genome and their Potential Utilization for Comparative Genomics in Cruciferae

We evaluated the nature of microsatellites in Brassica rapa in order to develop an informative and reliable DNA marker system for Brassica genetic analysis. Microsatellites were isolated by hybridization screening of an unconcentrated small-insert genomic library using tri-and dinucleotide probes. Of 45,000 clones screened, 210 had repeat sequences, in which 228 microsatellites were identified. The most frequent microsatellite motif was (GA)n at a frequency of one every 4.8 × 105 bp, followed by (CAA)n at one every 5.0 × 105 bp. The frequency of the tri- and dinucleotide microsatellites throughout the B. rapa genome was estimated to be one every 120 Kb. The number of repeats and the polymorphism information content of the dinucleotide microsatellites were higher than those of the trinucleotide microsatellites. More than 90 % of the primer pairs successfully amplified the corresponding microsatellite regions in other Brassica species. Furthermore, a considerable portion of them could be used in other Cruciferous species, 78.5 % in Raphanus sativus, 68.6 % in Sinapis alba and 39.8 % in Arabidopsis thaliana. Based on these results, we concluded that B. rapa microsatellites have a high potential for the development of DNA markers that could contribute to the genetic analysis of Brassica and other Cruciferae.

Isolation, characterization and cross‐species amplification of eight microsatellite DNA loci in the migratory locust (Locusta migratoria)

AbstractEight polymorphic di‐ and trinucleotide microsatellite loci suitable for population genetic analysis were developed in Locusta migratoria from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.45 to 0.97, with the observed allele numbers varying between nine and 45. The overall microsatellite cloning efficiency in L. migratoria is 14%, suggesting that in migratory locusts, microsatellite sequences are abundant and should provide a valuable and easily accessible source of nuclear markers for genetic studies. These microsatellite loci were highly Locusta‐specific, with only very limited cross‐species applicability.

POPULATION GENETIC STRUCTURE OF THE AFRICAN MALARIA MOSQUITO ANOPHELES FUNESTUS IN KENYA

Anopheles funestus Giles is a major malaria vector in Africa, but little is known about the genetic structure of natural populations. In this study, microsatellite markers were used to investigate the genetic structure of A. funestus populations from Kenya. Two populations from western Kenya 80 km apart and two from coastal Kenya 50 km apart were collected and examined for allelic variation at five trinucleotide microsatellite loci. We found A. funestus Giles was the predominant species (> 98%) in the A. funestus group in these populations. The western Kenya populations exhibited higher genetic diversity than the coastal populations. No significant genetic structure for populations within the coastal or western Kenya regions was detected. However, population genetic differentiation between the two regions was high (F(ST) = 0.208, R(ST) = 0.158), approximately two-fold higher than A. gambiae populations from the same area. The results suggest that the minimum area associated with a deme of A. funestus in western or coastal Kenya is larger than 50 km in diameter. The Great Rift Valley in east Africa, high-elevation mountains in western Kenya, and the vast arid area east to the Great Rift Valley may all play a role in restricting A. funestus gene flow between coastal and western Kenya.