Dinucleotide Microsatellite Loci Research Articles

PSX-30 Late-Breaking Abstract: Genotypes database of pedigree cattle population in Kazakhstan and the introduction of a calculating mechanism for livestock parentage verification

Abstract A significant increase in pedigree cattle in Kazakhstan has led to scientific work on the conservation of breed genotypes in Kazakhstan and the development of imported breeds. Aim of work: creation a national database and collection of genotyped DNA samples of cattle in Kazakhstan and the introduction of an automated statistical and mathematical calculation of the local pedigree cattle parentage verification. The methodology of work has been the selection of biological samples and registration of data on the animal. DNA and STR locus testing have been performed at the University of Queensland’s Animal Genetics Laboratory (Australia). DNA genotyping was carried out using 21 polymorphic dinucleotide microsatellite loci. The database of DNA genotypes of breeding animals has been written and introduced into the database of breeding livestock in Kazakhstan (www.plem.kz). The method of determining kinship and confirming the origin of the animal was implemented by calculating the probability of random coincidence of genetic markers, taking into account their distribution in the population according to the “product rule.” The frequency distribution of homozygous alleles for 12 microsatellite loci (table 1) recommended by ICAR and 21 DNA loci (table 2) in the context of breeds was calculated, which indicates an increase in reliability when calculating the origin of livestock, with an increase in the number of microsatellite loci studied to 21 markers. The lower probability threshold is established, with 95% statistical significance confirms the relationship between the compared animals (table 3). A database of genotyped DNA samples of beef and dairy cattle breeds in Kazakhstan has been created and is currently used in pedigree beef cattle breeding. Access to the DNA database is provided to Kazakhstan genetic laboratories, and an automated calculation of the mathematical probability of parentage verification for a breeding animal and the generation of a genetic certificate was introduced on the website www.plem.kz

Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may...

Multiple paternity at the largest green turtle (Chelonia mydas) rookery in the Mediterranean

Sea turtle abundance estimates are often based on nest numbers or female-tagging projects. However, because male sea turtles do not emerge on the beach and are difficult to observe at sea, the number of males contributing to the breeding population is difficult to characterize. Using a molecular approach like genotyping hatchings to infer paternal genotypes can provide an indirect estimate of breeding male population size. This would help to estimate operational sex ratios, which is of vital significance for the conservation of sea turtle populations. We investigated the pattern of multiple paternity and reconstructed paternal genotypes in the largest green turtle nesting site of the Mediterranean, Akyatan beach, Turkey, using nine dinucleotide microsatellite loci. We genotyped 22 randomly selected unique female green turtles (approx.33.3% of all nesting females during 2009 nesting season) and tissue biopsies from 15 hatchlings from each of their nests n = 330). We identified multiple paternity in 59–63.6 % of the nests analysed and identified 68 individual males that had sired offspring within these nests. The operational sex ratio within our sample of the population was 1 female for every 3 males. We found 3 repeating males (males that sired offspring with more than one female) in 6 clutches resulting in 11 paternal sibships.

Lineage analysis by microsatellite loci deep sequencing in mice.

Lineage analysis is the identification of all the progeny of a single progenitor cell, and has become particularly useful for studying developmental processes and cancer biology. Here, we propose a novel and effective method for lineage analysis that combines sequence capture and next-generation sequencing technology. Genome-wide mononucleotide and dinucleotide microsatellite loci in eight samples from two mice were identified and used to construct phylogenetic trees based on somatic indel mutations at these loci, which were unique enough to distinguish and parse samples from different mice into different groups along the lineage tree. For example, biopsies from the liver and stomach, which originate from the endoderm, were located in the same clade, while samples in kidney, which originate from the mesoderm, were located in another clade. Yet, tissue with a common developmental origin may still contain cells of a mixed ancestry. This genome-wide approach thus provides a non-invasive lineage analysis method based on mutations that accumulate in the genomes of opaque multicellular organism somatic cells. Mol. Reprod. Dev. 83: 387-391, 2016. © 2016 Wiley Periodicals, Inc.

ISOLATION AND CHARACTERIZATION OF 15 MICROSATELLITE LOCI FOR Ophicephalus argus Cantor.

The isolation and development of 15 polymorphic dinucleotide microsatellite loci were described for Ophicephalus argus from the Huaihe River. All loci were polymorphic in the 30 individuals tested. The number of alleles per variable locus ranged from nine to seventeen, with a mean of 12.00. These novel microsatellite loci showed high level of polymorphism. Observed and expected heterozygosities ranged from 0.793 to 0.929 and from 0.841 to 0.952, respectively. Two loci were found deviated from HWE in the sampled population after Bonferroni correction. These microsatellite loci will be useful for revealing population structure, genetic diversity, and phylogeography of Ophicephalus argus.

Isolation and characterization of thirteen novel dinucleotide microsatellite loci from Tetraena mongolica Maxim

We developed 13 polymorphic microsatellite loci isolated from Tetraena mongolica genomic libraries, from a small area in western Inner Mongolia, China. Polymorphisms detected in 18 individuals ranged from 3 to 18 alleles per locus. The observed and expected heterozygosity ranged from 0.444 to 0.944 and 0.586 to 0.948, respectively. The polymorphic information content value ranged from about 0.505 to 0.916. Three loci showed significant deviations from the Hardy–Weinberg equilibrium, while we did not detect any linkage between loci. These markers show high levels of polymorphism, and are of use for future studies.

In silico mining of microsatellites in coding sequences of the date palm (Arecaceae) genome, characterization, and transferability.

• Premise of the study: To complement existing sets of primarily dinucleotide microsatellite loci from noncoding sequences of date palm, we developed primers for tri- and hexanucleotide microsatellite loci identified within genes. Due to their conserved genomic locations, the primers should be useful in other palm taxa, and their utility was tested in seven other Phoenix species and in Chamaerops, Livistona, and Hyphaene.• Methods and Results: Tandem repeat motifs of 3–6 bp were searched using a simple sequence repeat (SSR)–pipeline package in coding portions of the date palm draft genome sequence. Fifteen loci produced highly consistent amplification, intraspecific polymorphisms, and stepwise mutation patterns.• Conclusions: These microsatellite loci showed sufficient levels of variability and transferability to make them useful for population genetic, selection signature, and interspecific gene flow studies in Phoenix and other Coryphoideae genera.

Genetic structure and admixture between the Posavina and Croatian Coldblood in contrast to Lipizzan horse from Croatia

The Posavina and Croatian Coldblood are Croatian autochthonous horse breeds with interwoven breeding histories for which studbooks have only recently been established. The Lipizzan breed has the oldest formalized breeding and no record of recent genetic introgression from other breeds in Croatia. We analyzed the genetic structure, interbreeding, and breed characteristics by genotyping nine dinucleotide microsatellite loci for 53 Posavina, 37 Croatian Coldblood, and 33 Lipizzan horses and showed that differing breeding schemes and histories have had a strong and measurable impact on the population genetic structure within and between the three breeds. A Bayesian clustering method demonstrated that two population clusters best explain the genetic structure. Samples from the pre-defined breeds of the Posavina and Croatian Coldblood were assigned to a separate genetic cluster, while Lipizzan specimens were assigned to another distinct genetic group. Twelve samples of the Posavina/Croatian Coldblood cluster (13%) showed admixed ancestry with Lipizzan horses. A test for heterozygosity excess, allele frequency distribution mode-shift, and M-ratio test were used to detect genetic evidence of recent population bottlenecks, none of which provided evidence for bottlenecks in the Posavina and Croatian Coldblood populations. In contrast, although somewhat ambiguous, evidence suggests a genetic bottleneck in the Lipizzan population in Croatia.

Isolation of microsatellite loci for the predatory fish Hoplias microlepis (Characiformes: Erythrinidae) from a highly impacted river system in western Ecuador

We developed 14 polymorphic, dinucleotide microsatellite loci for Hoplias microlepis, a large predatory fish from a highly impacted river in western Ecuador. These were isolated using a next-generation pyrosequencing approach and tested in a sample of 32 fish. None of the loci were in linkage disequilibrium and all appeared to be in Hardy–Weinberg. Nine of the loci exhibited greater variability, having 3–8 alleles (average = 5.22), observed heterozygosities between 0.22 and 0.81 (average = 0.58), and expected heterozygosities between 0.20 and 0.83 (average = 0.56). The other five loci had 2 alleles and heterozygosity values generally close to 0. This is the first study dedicated to isolating microsatellites for a freshwater fish from the highly impacted aquatic ecosystems of western Ecuador.

Isolation and characterization of microsatellite markers for Thujopsis dolabrata var. hondai (Cupressaceae)1

Microsatellite markers were developed for the endemic Japanese species Thujopsis dolabrata var. hondai for studies on forest ecology and tree breeding. We characterized 10 dinucleotide microsatellite loci by screening primers developed using a simple sequence repeat-enriched library. The number of alleles per locus ranged from eight to 44 with an average of 20.3. The observed and expected heterozygosities ranged from 0.326 to 0.854 and from 0.670 to 0.976, respectively. The development of these markers can be used to assess useful genetic information for ecological studies and tree breeding in T. dolabrata var. hondai.

Development and characterization of nine microsatellite loci from the Korean hare (Lepus coreanus) and genetic diversity in South Korea

The Korean hare, Lepus coreanus, is an important mammal in ecosystem food chains, and is distributed across the entire Korean peninsula and northeastern China. Polymorphic microsatellite loci were developed using the biotin-enrichment technique for use in population genetics studies. Five trinucleotide and four dinucleotide microsatellite loci were selected and tested on 22 Korean hare specimens collected from Gangwon Province and Gyeongsangbuk Province in South Korea. The number of alleles across the two sampling regions ranged from three to nine with a mean of 6.1. Mean observed and expected heterozygosities and polymorphic information content were 0.540, 0.627 and 0.579, respectively. Only one locus, Lc06, showed departure from Hardy-Weinberg equilibrium after applying the Bonferroni correction. Four microsatellites, Lc01, Lc03, Lc12, and Lc19, satisfied the criteria to serve as a core set of markers recommended for population genetics studies. These new microsatellite markers will be widely applicable to future genetic studies for management and conservation of the Korean hare and related species, including assessment of the genetic diversity and population structure of L. coreanus.

Development of 16 microsatellite loci of the tropical sea cucumber Stichopus horrens with cross-amplification in the congeneric species Stichopus monotuberculatus

This study employed the enriched genomic library method to isolate and characterize dinucleotide microsatellite loci in the tropical sea cucumber species Stichopus horrens. Sixteen novel microsatellite loci were identified and we assessed the polymorphism of 40 individual samples from Hainan Province, China. The numbers of alleles ranged from 8 to 18 (total 213, mean 13.3), the observed and expected heterozygosity varied from 0.294 to 0.971, and from 0.720 to 0.913, and 12 of the 16 loci were found to be in accordance with the Hardy–Weinberg equilibrium. In addition, the successful cross-amplification of 8 loci with the congeneric Stichopus monotuberculatus, suggests that these markers could also be used in this species. These polymorphic markers can be useful for additional studies of population structure and stock management of S. horrens and related species.

Isolation and characterization of 20 new microsatellite loci in Coriaria nepalensis (Coriariaceae)

Microsatellite markers were developed for Coriaria nepalensis, a traditional Chinese medicinal and ornamental plant, to investigate its genetic diversity and population genetic structure as well as its evolutionary history. Twenty-eight dinucleotide microsatellite loci were identified in C. nepalensis, 20 of which showed polymorphism among five populations. The expected heterozygosities were 0-1 (mean 0.469). These markers may be useful for further investigation of the population genetics, systematics, and phylogeography of Coriaria nepalensis.

Isolation and characterization of microsatellite loci in Schoenoplectus juncoides (Cyperaceae)1

We developed microsatellite markers for Schoenoplectus juncoides, a weed found in rice paddy fields in eastern Asia. Ten dinucleotide microsatellite loci were isolated, and genetic variability within and among local populations were characterized. The number of alleles per locus ranged from 2 to 6 with a mean of 3.6. Expected heterozygosity per locus ranged from 0.067 to 0.471, whereas observed heterozygosity showed much lower values ranging from 0.000 to 0.083. Most of the loci showed significant deviation from Hardy-Weinberg equilibrium and high inbreeding coefficient values (0.525-1.000). All the 10 microsatellite loci displayed polymorphism within and among populations. These loci are useful genetic markers for microevolution and ecological studies of S. juncoides populations.

Characterization of Polymorphic Microsatellites in the Giant Bulldog Ant,Myrmecia brevinodaand the Jumper Ant,M. pilosula

The ant genus Myrmecia Fabricius (Hymenoptera: Formicidae) is endemic to Australia and New Caledonia, and has retained many biological traits that are considered to be basal in the family Formicidae. Here, a set of 16 dinucleotide microsatellite loci were studied that are polymorphic in at least one of the two species of the genus: the giant bulldog ant, M. brevinoda Forel, and the jumper ant, M. pilosula Smith; 13 are novel loci and 3 are loci previously published for the genus Nothomyrmecia Clark (Hymenoptera: Formicidae). In M. brevinoda, the total of 12 polymorphic microsatellites yielded a total of 125 alleles, ranging from 3 to 18 with an average of 10.42 per locus; the observed and expected heterozygosities ranged from 0.4000 to 0.9000 and from 0.5413 to 0.9200, respectively. In M. pilosula, the 9 polymorphic loci yielded a total of 67 alleles, ranging from 3 to 12 with an average of 7.44 per locus; the observed and expected heterozygosities ranged from 0.5625 to 0.9375 and from 0.4863 to 0.8711, respectively. Five loci were polymorphic in both target species. In addition, 15 out of the 16 loci were successfully amplified in M. pyriformis. These informative microsatellite loci provide a powerful tool for investigating the population and colony genetic structure of M. brevinoda and M. pilosula, and may also be applicable to a range of congeners considering the relatively distant phylogenetic relatedness between M. pilosula and the other two species within the genus Myrmecia.

Isolation and characterization of 50 nuclear microsatellite markers for Cathaya argyrophylla, a Chinese endemic conifer

Microsatellite primers were developed for the endangered Cathaya argyrophylla (Pinaceae) to investigate its genetic diversity and population genetic structure, as well as its evolutionary history. • Fifty dinucleotide microsatellite loci were identified in two populations. The number of alleles per locus ranged from 1 to 6, with a mean of 2.84. The observed and expected heterozygosities ranged from 0 to 0.889 and from 0 to 0.779, respectively. • These markers will facilitate further studies on the population genetics and evolutionary history of Cathaya argyrophylla.

High genetic diversity and possible evidence of a recent bottleneck in Adriatic bottlenose dolphins ( Tursiops truncatus)

The bottlenose dolphin ( Tursiops truncatus) is the only resident marine mammal species in the Croatian part of the Adriatic Sea, with an estimated number at around 220 individuals. It is an endangered and legally protected species in Croatia, and its demographic history is poorly known. This study investigates the level of genetic diversity in the bottlenose dolphin population from the Croatian part of the Adriatic Sea and a possibility of recent population size contraction, since there are indications that there has been intensive eradication operations in the mid 20th century that might have caused reductions in the effective population size and might have resulted in a loss of genetic variation. Thirty samples were genotyped at 12 dinucleotide microsatellite loci. The mean allelic richness (6.835 ± 0.705) and mean expected heterozygosity (0.692 ± 0.05) revealed high level of genetic diversity. Bottleneck analysis gave ambiguous evidence for a recent population decline in the investigated bottlenose dolphin population. The M ratio test, with two sets of parameter values, suggested a recent bottleneck; whereas the analysis by the Bottleneck program under three mutation models (TPM, SMM and IAM) showed no evidence for a genetic bottleneck. We take a more conservative approach to the interpretation of these results by accepting the evidence of a recent bottleneck. We suggest maintaining the current level of bottlenose dolphin protection in the area and careful monitoring of the population in the future.

Microsatellite markers for mating system and population analyses of the spectacled caiman Caiman crocodilus (Linnaeus 1758)

We used a microsatellite enriched library to isolate and characterize 12 di-nucleotide microsatellite loci of Caiman crocodilus. Microsatellites were characterized in populations of C. c. crocodilus from Piagacu-Purus, Amazonas, Brazil and C. c. yacare from Caceres, Mato Grosso, Brazil. Results of characterization were comparable between the two subspecies, with number of alleles varying from three to 20 and one to 14 per locus in C. c. crocodilus and C. c. yacare, respectively. Observed heterozygosities ranged from 0.088 to 0.816 and 0.115 to 0.833, respectively. Joint paternity exclusion (QC) was >0.999 in both subspecies, and probability of genetic identity varied from 4.631 × 10−13 in C. c. crocodilus to 2.233 × 10−8 in C. c. yacare. All loci are unlinked, and with the exception of three loci in C. c. crocodilus all loci were at Hardy–Weinberg equilibrium. The characteristics of these loci makes them an excellent tool set for the study of mating systems, and fine-scaled population structuring in the Caiman crocodilus species complex.

Polymorphic microsatellite markers for the overexploited Amazonian fish, Semaprochilodus insignis (Jardine and Schomburgk 1841)

We used a microsatellite-enriched library to isolate and characterize 16 di-nucleotide microsatellite loci in Semaprochilodus insignis. Microsatellites were characterized in a population sample from Piagacu-Purus (Purus River, Amazonas, Brazil) and in eight individuals each of S. brama from Itaipavas (Araguaia River, Para, Brazil), S. taeniurus from Novo Airao (Negro River, Amazonas, Brazil), S. kneri (Caura River, Venezuela), and Prochilodus nigricans also from Piagacu-Purus. In S. insignis observed heterozygosities ranged from 0.516 to 0.968, expected heterozygosities from 0.574 to 0.973, and the number of alleles per locus varied from 4 to 32 in 31 analyzed individuals. All loci were unlinked and three loci showed significant deviations from Hardy–Weinberg equilibrium. Most markers could be successfully transferred to the other four prochilodontids, making them potentially useful for conservation genetic studies of Semaprochilodus spp.

Isolation and characterization of twenty dinucleotide microsatellite loci in the buff-throated partridge, Tetraophasis szechenyii

Twenty dinucleotide microsatellite loci were isolated from AC-enriched libraries of the buff-throated partridge, Tetraophasis szechenyii. These loci were tested on 25 individuals of T. szechenyii, and most loci are proved to be highly polymorphic. The number of alleles per locus ranged from three to eight. Polymorphism information content (PIC) ranged from 0.552 to 0.838. Observed and expected heterozygosities (Ho, He) were varied 0.480–0.792 and 0.633–0.872, respectively. The newly identified polymorphic markers will facilitate the study of T. szechenyii on the population genetics and genetic linkage map construction.