Amplification Of Microsatellite Loci Research Articles

Development and multiplexing of microsatellite markers using pyrosequencing in a tetraploid plant, Vaccinium uliginosum (Ericaceae)

Objective: Background and aims - We developed a new set of microsatellite primers in the tetraploid perennialshrub, Vaccinium uliginosum L., to investigate genetic diversity and population genetic structure.Methods - Using pyrosequencing, we identifi{ligature}ed and designed primers for PCR amplification ofmicrosatellite loci in V. uliginosum. The primers were first screened for amplification and polymorphismby PCR and agarose gel electrophoresis. PCR products of selected primers were then ligated into avector, amplified with a universal fluorescently labelled forward primer and a specific reverse primer, andelectrophoresed on a capillary sequencer to check the scorability of the peaks. Finally, multiplexes weredesigned and tested on ninety individuals sampled in three Belgian populations.Key results - We designed and tested a total of 52 primer pairs, of which nine yielded scorable peaks, i.e.eight di- and one tri-nucleotide loci with seven to fourteen alleles per locus in three Belgian populations. Theexpected heterozygosity was high, ranging from 0.52 to 0.87 (mean = 0.77). Genetic diversity (Shannon'sdiversity, H') ranged from 1.27 to 1.42 and was much higher than that observed by Albert et al. (2005)using RAPD-analyses in the same populations. This could be due to the higher polymorphism retrievedwith microsatellite markers.Conclusions - The microsatellite markers we developed showed enough polymorphism to investigategenetic diversity and structure even at small spatial scales, gene and pollen dispersal (through paternityinference) or outcrossing rates. © 2014 Botanic Garden Meise and Royal Botanical Society of Belgium.

Development of novel, cross-species microsatellite markers for Acropora corals using next-generation sequencing technology

The genus Acropora (Scleractinia, Acroporidae) is one of the most widespread coral genera, comprising the largest number of extant species among scleractinian (reef-building) corals. Molecular phylogenetic studies have suggested that A. tenuis belongs to the most basal clade (clade I) while A. digitifera belongs to a derived clade (clade IV). In order to develop microsatellite markers that would be useful for most Acropora species, we sequenced the genomic DNA of A. tenuis, using a next generation sequencer (Illumina MiSeq), and designed primer sets that amplify microsatellite loci. Afterward we selected primer pairs with perfectly matched nucleotide sequences from which at least one primer was uniquely mapped to the A. digitifera genome. Fourteen microsatellite markers showed non-significant departure from Hardy–Weinberg equilibrium (HWE) in both A. tenuis and A. digitifera. Thus these markers could be used for wide range of species and may provide powerful tools for population genetics studies and conservation of Acropora corals.

Microsatellites facilitate species delimitation in Congolese Ancistrocladus (Ancistrocladaceae), a genus with pharmacologically potent naphthylisoquinoline alkaloids

AbstractAncistrocladus (Ancistrocladaceae) is an Old World genus of woody lianas, occurring in tropical Africa and Southeast Asia. Its species produce a number of structurally unique naphthylisoquinoline alkaloids with promising activities against the pathogens of tropical infectious diseases and against HIV. Species identification for phytochemical and pharmacological research is difficult because diagnostic reproductive features are frequently not available. Using enrichment and subcloning, and a 454 shotgun sequencing technique with semi‐automatized SSR motif screening, we have successfully developed a set of primers that amplify microsatellite loci across most Ancistrocladus species. The primers are designed to support species identification of indeterminate samples. Twelve markers from a total of 99 tested loci were identified as suitable for cross‐species amplification. The markers produced distinct species clusters in a STRUCTURE analysis and a principal coordinate analysis (PCoA) based on a test sampling consisting of 21 samples from six different Ancistrocladus species. When applied to 74 samples of Ancistrocladus congolensis s.l., a complex of morphologically similar species from tropical Central Africa, these markers showed medium to high variability in ten populations. A STRUCTURE analysis of the A. congolensis s.l. samples revealed five distinct clusters, confirming three of the five taxonomically recognized units and also indicating the presence of a hitherto unrecognized species in the region. The taxonomic status of the recently described new species A. ileboensis is supported by our results. The novel microsatellite markers developed in this study enable rapid screening of samples and provide an efficient tool for species identification and detection of cryptic taxa.

Microsatellite DNA markers in Shorea platyclados (Dipterocarpaceae): genetic diversity, size homoplasy and mother trees

Cross-specific amplification of microsatellite loci greatly enhances the effectiveness of this marker system. This shortcut would greatly enhance our examination of the gene flow and population structure of trees in diverse tropical rainforests. To explore the effectiveness and limitations of this approach, we examined allelic diversity at six microsatellite loci, originally developed in a congeneric species, in three populations of Shorea platyclados from Peninsular Malaysia. Fragment sizing was performed by an efficient and sensitive (1 bp resolution) technique using capillary electrophoresis, ethidium bromide detection, and minimal clean-up. Fragment size ranges were conserved between species and null allele frequencies were low. Higher overall levels of genetic diversity were detected in our study. Variation among populations was directly related to geographic distance. Fragment size class distributions suggest that each locus should be studied using different evolutionary models. Direct sequencing of SSR fragments revealed that size differences were due to changes in both the flanking regions and repeat motifs. Several clear examples of size homoplasy were observed, along with the disruption of perfect repeats, suggesting that cross-specific amplification of microsatellite loci requires an additional level of confirmation at the DNA sequence level before the influence of size homoplasy and changes in repeat structure can be assessed. Simulation studies demonstrate that the increasing intensity of timber harvest leads to higher variability in levels of potential heterozygosity and decreasing total number of alleles in the remnant "mother trees" The careful selection of "mother" trees can greatly enhance the future genetic diversity of populations.   

Development and cross-species amplification of microsatellite loci for Puccinellia maritima, an important engineer saltmarsh species.

The grass Puccinellia maritima is an important saltmarsh ecosystem engineer exhibiting wide morphological variation, which is partially genetically determined. Nevertheless, nothing is known about its population genetics or how neutral genetic variation is distributed throughout its geographical range. Here, we describe 12 polymorphic microsatellites pooled into two multiplexes for this octoploid species. Assessment of 24 samples from three populations revealed 4 to 29 alleles per locus, with variation in allele presence and abundance between populations. The transferability of these markers is reported based on their cross-amplification in six other Puccinellia species of different ploidy levels.

Analysis of the Microsatellite Variation in the Common Hybrid Between Russian Sturgeon (Acipenser Gueldenstaedtii Brandt and Ratzeburg, 1833) and Siberian Sturgeon (Acipenser Baerii Brandt, 1869) from Aquaculture

ABSTRACT Sturgeons such as Acipenser baerii and Acipenser gueldenstaedtii are the most common species farm raised worldwide in aquaculture, because of the dwindling natural sources of caviar and meat. Also, these species can easily participate in the formation of an intraspecific hybrid with a great potential for growth in aquaculture. Microsatellites are nuclear markers consisting of short repetitive sequence, dispersed across the entire genome with characteristics such as relatively small size and high level of polymorphism. The aims of the present study were to optimize a protocol for microsatellite multiplexing and analysis of genetic diversity in hybrid sturgeons farmed in Romania. Genomic DNA was isolated from fins, and four pairs of primers were designed to amplify microsatellite loci: LS 19, LS 68, Aox 9, and Aox 45. Amplification of the microsatellite loci was carried out in one 3-Plex reaction for LS 19, LS 68, and Aox 9, and monoplex reaction for Aox 45. For an individual locus we obtained four alleles for Aox 45, eleven alleles for Aox 9, six alleles for LS 68, and eight alleles for LS 19. The results will be applied to test the broodstocks at Romanian hatcheries and to increase the efficiency of breeding.

Cross-species amplification of microsatellite loci developed for Passiflora edulis Sims. in related Passiflora Species

The aim of this study was to evaluate the selected 41 SSR markers developed for yellow passion fruit (Passiflora edulis f. flavicarpa Sims.) for their transferability to 11 different Passiflora species. Twenty-one SSR were successfully amplified in 10 wild species of passion fruit producing 101 bands. All the markers were amplifiable for at least one species. The mean transferability was 68,8%, ranging from 15,4% (primer PE11) to 100 % (PE13, PE18, PE37, PE41 and PE88). Transferability was higher for the species from the Passiflora subgenus than for those from the Decaloba and Dysosmia subgenus. The results indicated a high level of nucleotide sequence conservation of the primer regions in the species evaluated, and consequently, they could potentially be used for the establishment of molecular strategies for use in passion fruit breeding and genetics.

Development of microsatellite markers for Leucosceptrum japonicum and L. stellipilum (Lamiaceae).

• Premise of the study: We developed 10 new primers for amplifying microsatellite loci for both Leucosceptrum japonicum and L. stellipilum to study the genetic structure of hybrid zones between these two species.• Methods and Results: We isolated DNA fragments containing microsatellites from an enriched library and designed the 10 primer pairs amplifying the fragments. The number of alleles per locus ranged from one to 11 in both L. japonicum and L. stellipilum. The observed heterozygosity ranged from 0.00 to 0.84 and 0.00 to 1.00 in L. japonicum and L. stellipilum, respectively. The expected heterozygosity ranged from 0.00 to 0.84 and 0.00 to 0.89 in L. japonicum and L. stellipilum, respectively.• Conclusions: These newly developed markers will be useful in investigating genetic structure and hybridization patterns in hybrid zones between L. japonicum and L. stellipilum.

Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.MethodsImmunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).ResultsThe positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.ConclusionsOur study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.

Molecular ecology of the Neotropical otter (Lontra longicaudis): non-invasive sampling yields insights into local population dynamics

Non-invasive genetic analysis has been frequently employed to estimate ecological and population parameters for many secretive and/or threatened species. However, Neotropical carnivores have so far been scarcely targeted by such studies. The Neotropical otter (Lontra longicaudis) is a poorly-known species for which local levels of genetic diversity and demographic parameters are virtually absent. We employed non-invasive sampling and amplification of microsatellite loci to investigate population size and density, spatial organization, and relatedness of a wild Neotropical otter population in an Atlantic forest area in southern Brazil. We directly identified 28 individuals and estimate a rather high population density at the study site. Spatial organization analysis indicated that male cumulative displacement was higher than that of females, with the latter sex showing evidence of philopatric behaviour. Also, the reconstruction of genealogical relationships suggests that spatial organization in this otter appears to be influenced by relatedness. By allowing the testing of specific hypothesis targeting these issues, our results provided important glimpses into the Neotropical otter's population biology. Moreover, the findings of the present study reaffirm the power of non-invasive genetics to investigate the biology of this elusive species, and open up new avenues for ecological and demographic studies of other Neotropical carnivores. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 932–948.

Molecular data contradicts historical records and cautions translocation of the Lord Howe Island masked owl

Masked owls, reputedly all of the Tasmanian race (Tyto novaehollandiae castanops) were introduced onto Lord Howe Island (LHI) in the 1920s in an attempt to control the black rat (Rattus rattus). This attempt, however, has been unsuccessful and a co-eradication of the rats and masked owls has been planned to reduce the threat to endemic species and breeding seabirds on the island. As the Tasmanian masked owl is considered endangered, translocation of LHI masked owls to Tasmania has been suggested. Before translocation is considered the ancestry of the LHI masked owl needs to be confirmed, as LHI masked owls are typically smaller and paler than individuals occurring in Tasmania. Here we sequenced three sections of mitochondrial gene regions: cytochrome b, ATP6 and ND3 to assess the provenance of the LHI masked owl and screened a suite of microsatellite loci isolated from the barn owl (Tyto alba) to assess contemporary divergence. Phylogenetic analysis revealed two clades, one exhibited by individuals from LHI and south-eastern mainland Australia and the second by those from Tasmania. Cross species amplification of microsatellite loci was successful, with 18 loci polymorphic. Genotypic data revealed significant sub-structuring between LHI, south-eastern mainland Australia and Tasmania. Data presented here indicate that the south-eastern mainland masked owl was introduced to LHI and subsequently reproduced. The genetic integrity of the LHI masked owl population is therefore questionable and as such LHI individuals may not be suitable for translocation to Tasmania.

Short Communication Cross-species amplification of selected zebrafish, central stoneroller, and finescale dace microsatellites in lake minnow populations

Fifteen sets of PCR primers designed for the amplification of microsatellite loci from Danio rerio and Phoxinus neogaeus Campostoma DNA that have been proven applicable for molecular studies of several species of cyprinids were tested for amplification of microsatellites from lake minnow DNA. The samples were taken from 298 fish inhabiting 6 distinct populations located in Poland, and DNA was extracted from dried fin clips. There were 6 sets of primer loci that were identified as useful for amplification of microsatellites from lake minnow DNA, which were polymorphic and could be applied in population genetics of this species. Three other sets of primers provided PCR products with a considerable number of stutter bands obscuring the identity of true microsatellite alleles. The amplification of lake minnow microsatellites using the 6 remaining primer sets was unsuccessful.

Cross Species Amplification of Adzukibean Derived Microsatellite Loci and Diversity Analysis in Greengram and Related Vigna Species

Greengram or mungbean ( Vigna radiata (L.) Wilczek) is a well known grain legume in Asian countries. Among the different DNA markers, microsatellite or simple sequence repeats (SSRs) are the markers of choice for various genetic studies due to co-dominant nature, loci specificity and high reproducibility. In the present study, a set of thirty-five microsatellite primer pairs derived from adzukibean ( Vigna angularis (Willd.) Ohwi & Ohashi) were used to assess the transferability and tested for their ability to amplify microsatellite loci in greengram and related Vigna species. Of the thirty five microsatellite markers, thirty-two were successfully amplified across the thirty six genotypes and twenty eight were polymorphic. A total of 83 microsatellite alleles were generated with an average of 2.96 alleles per locus. Number of alleles ranged from two to five. Dendrogram formed based on UPGMA, 36 genotypes were grouped into five clusters. Similarly, the neighbour-joining tree developed based on weighted average for dissimilarity matrix grouped 36 genotypes into five groups. The finding suggests that adzukibean derived microsatellite markers are highly informative and could be used to improve the greengram at molecular level.

Identification and amplification of microsatellite loci in deep-sea tubeworms of the genus Escarpia (Polychaeta, Siboglinidae)

Deep-sea cold seeps support diverse communities that include vestimentiferan tubeworms of the genus Escarpia. To investigate population structure to understand how deep-sea communities are established, several polymorphic microsatellites were developed using 454 pyrosequencing. Eleven and sixteen loci were identified in Escarpia laminata (n = 129) and Escarpia southwardae (n = 80). Additionally, the amplification of microsatellites is expected to be successful in closely related species and can help resolve their phylogenetic relationships. Nine of the E. laminata loci were amplified across three Escarpia species (n = 229). Four of the twenty-eight loci deviated from Hardy–Weinburg Equilibrium and no linkage disequilibrium was found in the loci tested here.

Genetic diversity in Populus nigra plantations from west of Iran.

In order to adopt strategies for forest conservation and development, it is necessary to estimate the amount and distribution of genetic diversity in existing populations of poplar in Iran. In this study, the genetic diversity between eight stands of Populus nigra established in Kermanshah province was evaluated on the basis of molecular and morphological markers. To amplify microsatellite loci (WPMS09, WPMS16 and WPMS18), DNA extraction from young and fresh leaveswas done. Various conditions of the PCR assay were examined and to evaluate the morphological variation of the morphological characters leaves (consist of 19 traits) were measured. In addition, height growth was measured, to evaluate the growth function of the stands in homogeneous conditions. Genetic diversity in term of polymorphic loci was 0%, because three investigated microsatellite loci were monomorphic. The total number of alleles for 3 microsatellite loci was 6 (na = 2, ne = 2, heo = 1, hee = 0.51). Genetic identity based on Nei was 100%, so genetic distance was 0%. The whole sampled trees represented the same thus the genotype. No significant differences between the mean values of all morphological characters and height growth were revealed. Observed genetic similarity gave indication that same ramets had been selected to plant in poplar plantation established in Kermanshah province. These results suggest the need for an initial evaluation of the genetic diversity in selected ramets for planting in plantation to avoid repetition.

Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species

Studies of population genetics increasingly use next-generation DNA sequencing to identify microsatellite loci in nonmodel organisms. There are, however, relatively few studies that validate the feasibility of transitioning from marker development to experimental application across populations and species. North American coralsnakes of the Micrurus fulvius species complex occur in the United States and Mexico, and little is known about their population structure and phylogenetic relationships. This absence of information and population genetics markers is particularly concerning because they are highly venomous and have important implications on human health. To alleviate this problem in coralsnakes, we investigated the feasibility of using 454 shotgun sequences for microsatellite marker development. First, a genomic shotgun library from a single individual was sequenced (approximately 7.74 megabases; 26,831 reads) to identify potentially amplifiable microsatellite loci (PALs). We then hierarchically sampled 76 individuals from throughout the geographic distribution of the species complex and examined whether PALs were amplifiable and polymorphic. Approximately half of the loci tested were readily amplifiable from all individuals, and 80% of the loci tested for variation were variable and thus informative as population genetic markers. To evaluate the repetitive landscape characteristics across multiple snakes, we also compared microsatellite content between the coralsnake and two other previously sampled snakes, the venomous copperhead (Agkistrodon contortrix) and Burmese python (Python molurus).

Microsatellite loci to determine population structure in the yellow seahorse (Hippocampus kuda) and the three-spotted seahorse (H. trimaculatus)

Genetic diversity of seahorse species was studied using 12 polymorphic microsatellite DNA loci in the yellow seahorse Hippocampus kuda and the three-spotted seahorse H. trimaculatus from samples collected along the east and west coasts of India. Cross-species microsatellite loci amplification in populations of Indian seahorses showed significant numbers of private alleles associated with site-specific populations, which could be used for population genetic studies and management of species for conservation. Significant genotype heterogeneity indicated that the samples are not from the same gene pool, and that physical barriers exist which inhibit breeding between populations. The identified microsatellite loci can be further utilized in fine-scale population structure analysis of seahorses.

Characterization of nine microsatellite loci in the European polecat Mustela putorius

We developed a set of nine polymorphic microsatellite markers from a genomic library enriched for dinucleotide repeats in the European polecat Mustela putorius. Microsatellite loci amplification was tested on fresh tissues and museum samples collected over the last 40 years in central and northern Italy. Number of alleles per locus ranged from 3 to 7. Mean observed and expected heterozygosity were 0.48 and 0.61, respectively. This novel set of species-specific microsatellite loci will be particularly useful to assess fine patterns of genetic structure and degree of isolation of European polecat populations.

Screening of New Microsatellite DNA Markers from the Genome of Platyeriocheir formosa

The catadromous Platyeriocheir formosa is a crab endemic in Taiwan. To conserve P. formosa population diversity and ensure the sustainable use of this natural resource, we have developed new genetic markers, 17 polymorphic microsatellite loci, to promote the study of its population genetics in the future. In this study, more than 70 microsatellite sequences were found. Among these, 18 loci were selected to analyze the genetic diversity of P. formosa. With the exception of the Pfo15 locus, all of the remaining loci were polymorphic with allelic numbers ranging from 3–14. Heterozygosity within all 17 polymorphic loci ranged from 0.2–0.95 with an average of 0.55, which suggested that these loci are proper markers for studying population genetics. After we tested cross-specific amplification, eight and six primer sets could be successfully used for the amplification of microsatellite loci in morphologically similar Eriocheir sinensis and E. japonica, respectively; this suggests that they are useful markers for closely related species.

Low genetic diversity and evidence of population structure among subspecies of Nerodia harteri, a threatened water snake endemic to Texas

Nerodia harteri is a threatened small-bodied water snake that occupies one of the most restricted ranges of any snake within the continental United States. It is found closely associated with rivers and tributaries in the Colorado and Brazos river basins, which flow through north-central Texas. Nerodia harteri has been at the center of debate owing to conflicts between conservation efforts and the construction of dams that change or destroy its preferred habitat. Additionally, its taxonomic status has also been under contention with some authors recognizing two subspecies, the Brazos water snake (N. h. harteri) and the Concho water snake (N. h. paucimaculata), whereas other authors consider each separate species. Despite its relatively recent discovery during the 1940s, N. harteri has been the subject of several ecological studies, yet no population genetic assessment of either subspecies has been performed to date. We first evaluated the phylogenetic placement of both subspecies among other North American Nerodia using partial sequence data from the mitochondrial gene cytochrome-b. We then tested for population subdivision among four rivers encompassing the range of N. harteri and tested for the presence of admixture between river basins using mitochondrial sequence data (920 bp of cyt-b) and five cross-species amplified microsatellite loci. We found low mitochondrial haplotype diversity represented by two unique haplotypes in each river basin, which were separated by no more than four nucleotide changes. Nuclear loci showed low genetic diversity and population structuring within and among river basins. We did not find conclusive evidence of admixture between basins, and we support the presence of two separate evolutionarily significant units and two separate management units corresponding to each major river basin. Given increasing natural and anthropogenic threats, we recommend continued ecological and genetic monitoring of both subspecies.