Hardy-Weinberg Equilibrium Expectations Research Articles

Transcriptome sequencing and high‐resolution melt analysis advance single nucleotide polymorphism discovery in duplicated salmonids

Until recently, single nucleotide polymorphism (SNP) discovery in nonmodel organisms faced many challenges, often depending upon a targeted-gene approach and Sanger sequencing of many individuals. The advent of next-generation sequencing technologies has dramatically improved discovery, but validating and testing SNPs for use in population studies remain labour intensive. Here, we detail a SNP discovery and validation pipeline that incorporates 454 pyrosequencing, high-resolution melt analysis (HRMA) and 5' nuclease genotyping. We generated 4.59×10(8) bp of redundant sequence from transcriptomes of two individual chum salmon, a highly valued species across the Pacific Rim. Nearly 26000 putative SNPs were identified--some as heterozygotes and some as homozygous for different nucleotides in the two individuals. For validation, we selected 202 templates containing single putative SNPs and conducted HRMA on 10 individuals from each of 19 populations from across the species range. Finally, 5' nuclease genotyping validated 37 SNPs that conformed to Hardy-Weinberg equilibrium expectations. Putative SNPs expressed as heterozygotes in an ascertainment individual had more than twice the validation rate of those homozygous for different alleles in the two fish, suggesting that many of the latter may have been paralogous sequence variants. Overall, this validation rate of 37/202 suggests that we have found more than 4500 templates containing SNPs for use in this population set. We anticipate using this pipeline to significantly expand the number of SNPs available for the studies of population structure and mixture analyses as well as for the studies of adaptive genetic variation in nonmodel organisms.

Microevolutionary processes due to landscape features in the province of Jujuy (Argentina)

We seek to evaluate the influence of a diverse and rugged physical environment on the genetic background of human populations. We analyzed eight polymorphic Alu insertions in 226 individuals from Jujuy province (Argentina), which is composed of several regions with well-defined geographical features and marked contrasts between them associated with differences in altitude (range: 700-3300 m). This regional division was used to assess the spatial variation of the Alu diversity. Deviations from Hardy-Weinberg Equilibrium expectations resulting from heterozygous deficit were found for FXIIIB and PV92 in the highest subpopulations. Several Alu elements showed genetic heterogeneity between the highest region (La Puna) and the lowest regions (Valle and Selva). Similarly, a decreasing trend of the average heterozygosity according to altitude was found. Both the centroid method and the admixture analysis unveiled a gene flow above the average in lowland populations, indicating a higher proportion of foreign genes introduced by immigrants of European and African ancestry. Furthermore, several Alu frequency clines fitting the orientation of the altitude gradient were detected. Our study reveals a spatial patterning of the Alu diversity in Jujuy, most likely determined by disparities in landscape and environmental features between the different subregions. Differences in the physical environment would have drastically reduced the homogenizing effects of the gene flow and would have promoted genetic drift episodes in the highest subpopulations. Microevolutionary processes detected in Jujuy have played an important role in the shaping of the gene pool of the populations from this sub-Andean zone from Argentina.

Heterozygote deficiencies in parasite populations: an evaluation of interrelated hypotheses in the raccoon tick, Ixodes texanus

Population genetics is increasingly being used to study the biology of parasites at the scales of both the host (infrapopulation, IP) and host population (component population, CP). In this study we tested three mechanistic hypotheses that could explain deviations from Hardy-Weinberg equilibrium (HWE) expectations due to heterozygote deficits (HDs) at the CP scale in raccoon ticks (Ixodes texanus; n=718) collected from raccoons (Procyon lotor; n=91) and genotyped at 11 microsatellite loci. These hypotheses were presence of technical issues (for example, null alleles), hierarchical structure (for example, host demography) and cryptic structure (for example, kin structure). Although statistical support for null alleles existed, their presence would also be expected to lead to an underestimation in levels of relatedness, and thus kin structure. However, we found the opposite pattern: significant HD at the IP scale being more likely in CPs with significant vs non-significant levels of kin structure. Our analyses revealed that pooling of kin groups could lead to highly variable levels of F(IS) among loci, a pattern usually suggestive of null alleles. We used Monte-Carlo (MC) simulations to show that the existence of subdivided breeding groups and high variance in individual reproductive success could adequately explain deviations from HWE in I. texanus. Thus, our results indicate that biological factors can lead to patterns that have usually been interpreted as technical issues (for example, null alleles), and that it is important to take such factors into consideration because loci deviating from HWE likely reflect the effects of real biological processes.

Distribution of p53, GST, and MTHFR Polymorphisms and Risk of Cervical Intraepithelial Lesions in Sicily

Host factors, including genetic polymorphisms, may explain some of the individual differences in cervical cancer occurrence, and susceptibility information may be useful to address effective and specific preventive strategies for different countries. The purpose of the present study was to investigate the role of p53 codon 72, glutathione S-transferase class mu (GSTM1), glutathione S-transferase class theta (GSTT1), and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms on the risk for infection and/or of cervical intraepithelial lesions in women attending a colposcopy service in Catania, Sicily, with an already reported high prevalence of human papillomavirus. To identify the association among individual genetic polymorphisms, human papillomavirus infection, and histological findings, a case-control study was designed. Furthermore, to assess the combined effects of these polymorphisms on cervical cancer risk, combined genotype frequencies were compared among case patients and controls. Women homozygous for the p53 codon 72 Arg genotype were at a 5.6-fold higher risk for developing cervical intraepithelial neoplasia (CIN) 2 or 3 compared with those showing homozygosity for the Pro genotype or heterozygosity for the Pro/Arg genotype. The GSTM1 and GSTT1 null genotypes were overrepresented in infected patients and in women with CIN 2 or 3, although without any significant associations. A decreased risk for CIN of individuals homozygous for the MTHFR T allele was shown. After multiple logistic analyses, the presence of the allele 677T of the MTHFR gene was the best explaining protective factor against cervical carcinogenesis, and the allelic distribution in the control group followed the Hardy-Weinberg equilibrium expectations. However, the findings of our study still remain to be confirmed by additional and larger population-based surveys.

Autosomal STR allele frequencies and Y-STR and mtDNA haplotypes in Chilean sample populations

DNA from 1020 unrelated male individuals sampled from five locations of Chile (Iquique, Santiago, Concepción, Temuco, and Punta Arenas) were typed for autosomal STRs, Y-STRs, and the mtDNA Control Region. The populations were selected to develop reference databases to support forensic casework and relationship testing. Allele frequencies for 15 autosomal STR loci across the five sampled sites were compiled. As there was considerable overlapping of birthplaces of subjects sampled from these five sites, the pooled dataset was re-grouped based on birthplaces of the subjects into eight geo-political birthplace regions of the country. Each of these populations was evaluated for conformance to Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between loci and within the populations was assessed. Descriptive statistics, i.e., power of discrimination (PD), power of exclusion (PE), and mean power of exclusion were determined. No deviations from HWE expectations ( p < 0.05) and LD were detected. Combined PD and PE for each population exceeded 0.99999. Y-STR and mtDNA haplotype frequencies were developed and haplotype sharing within and between populations was evaluated. The PD for the Y-STR database is 0.99841 and for the mtDNA database it is 0.99356. Population substructure on the haplotype data evaluated by AMOVA indicated approximately 0.03% of the variation detected originated from differences among the eight birthplace regions. Independence between Y-STR haplotypes, mtDNA haplotypes, and autosomal loci was assessed using a mismatch distribution approach.

Isolation and characterization of microsatellite loci for mountain mullet (Agonostomus monticola)

We report on the isolation of 15 polymorphic microsatellite loci from mountain mullet (Agonostomus monticola). In the two populations sampled, loci exhibited two to 21 alleles and observed heterozygosity values ranged from 0.222 to 1.000. All loci conformed to Hardy-Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Although A. monticola is an important subsistence fishery in parts of its range, little is known about its ecology and many populations appear to be experiencing declines. These microsatellite loci should prove useful in the study of population structure of A. monticola and aid in other potential conservation efforts such as the management of hatchery broodstock.

Low Genetic Differentiation in the Populations of the Malabar Carp Labeo dussumieri as revealed by Allozymes, Microsatellites and RAPD

The population structure of Labeo dussumieri, an endangered and endemic cyprinid from three riverine locations in the Western Ghats, India was investigated using allozyme, microsatellite and RAPD markers. L. dussumieri samples were obtained from Meenachil, Manimala and Pamba River basins, Kerala. Fourteen (46.7%) out of 30 allozyme loci, seven microsatellite loci and 12 RAPD Operon decamers gave polymorphic pattern. Six allozyme loci (AAT-2*, EST-4*, GLDH*, GPI-2*, G6PDH* and LDH-2*) and three microsatellite loci (LdussG1, MFW19 and Bgon22) exhibited consistent significant deviation from Hardy-Weinberg Equilibrium expectations in different populations after probability level (P<0.05) was adjusted for sequential Bonferroni correction. All the three marker types demonstrated concordant results and various estimates revealed genetic variability within the subpopulations but surprisingly low level (= 0.0034 to 0.0081) of genetic differentiation among L. dussumieri from different river samples. AMOVA analysis also indicated low differentiation among subpopulations. No evidence for a recent genetic bottleneck was observed in L. dussumieri populations based on allozyme and microsatellite data set analysis. Meenachil, Manimala and Pamba Rivers open in to the southern end of Vembanad Lake in Kerala and are connected to each other in the lower reaches through an extensive network of natural canals. Common ancestry in the prehistoric period; and possible mixing of fish populations resulting in high gene flow across the rivers through the lake and interconnecting canals could have been responsible for the lack of significant allelic heterogeneity among the L. dussumieri populations. The stocks from the three rivers do not require different management strategies and for propagation assisted river ranching programme of this species, large effective breeding population can be developed by mixing individuals from three rivers

Genetic association study of selected candidate genes (ApoB, LPL, Leptin) and telomere length in obese and hypertensive individuals

BackgroundA genetic study was carried out among obese and hypertensive individuals from India to assess allelic association, if any, at three candidate loci: Apolipoprotein B (ApoB) minisatellite and two tetranucleotide repeat loci; LPL (Lipoprotein lipase) and Leptin. Attempt has also been made to find out whether telomere length attrition is associated with hypertension and obese individuals.MethodsVenous blood samples were collected from 37 normal, 35 obese and 47 hypertensive individuals. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) and PCR amplifications were achieved using locus specific primers. Genotyping of ApoB minisatellite was performed using 4% polyacrylamide gel electrophoresis (PAGE) followed by silver staining, whereas LPL and Leptin loci were genotyped using ALF Express™ DNA sequencer. Telomere length was determined using a recently developed real time based quantitative PCR, where the relative telomere length was determined by calculating the relative ratio of telomere (T) and single copy gene (S) PCR products which is expressed as T/S ratio.ResultsAll the three loci are highly polymorphic, display high heterozygosity and conform to Hardy-Weinberg's equilibrium expectations. ApoB minisatellite displayed 14 alleles, whereas LPL and Leptin tetranucleotide loci were having 9 and 17 alleles, respectively. Interestingly two new alleles (9 and 11 repeats) were detected at ApoB locus for the first time. The alleles at Leptin locus were classified as Class I (lower alleles: 149-200 bp) and Class II alleles (higher alleles: >217 bp). Higher alleles at ApoB (>39 repeats), predominant allele 9 at LPL and alleles 164 bp and 224 bp at Leptin loci have shown allelic association with hypertensive individuals. After adjusting the influence of age and gender, the analysis of co-variance (ANCOVA) revealed the relative telomere length (T/S ratio) in hypertensive individuals to be (1.01 ± 0.021), which was significantly different (P < 0.001) from obese (1.20 ± 0.023) and normal (1.22 ± 0.014) individuals. However, no significant difference in the relative telomere length was observed among male and female individuals, although age related decrease in telomere length was observed in these limited sample size.ConclusionThe present study revealed that allelic association at ApoB, LPL, Leptin loci and loss of telomere length may have strong genetic association with hypertensive individuals. However, further study on larger sample size is needed to draw firm conclusions.

An X-chromosome pentaplex in two linkage groups: Haplotype data in Alagoas and Rio de Janeiro populations from Brazil

Genotype data were obtained for five X-Chr STRs (DXS10079, DXS10074, DXS10075, DXS7424 and DXS101) in two different populations from Brazil, namely Alagoas and Rio de Janeiro. Observed genotype distributions in female samples for each locus do not show deviations from Hardy–Weinberg equilibrium expectations. Gametic association was tested for all pairs of loci in male samples. Significant association values were found between all pairs including DXS10079, DXS10074 and DXS10075, as well as between DXS7424 and DXS101, proving that these two groups of markers must be treated as haplotypes. No significant association could be found between markers from the two groups (DXS10079, DXS10074 or DXS10075 vs. DXS7424 or DXS101), although distances between them varied from 24 to 25 cM. When comparing haplotype frequencies in Alagoas, Rio de Janeiro, Germany and Ghana, significant differences were found between the Brazilian and the Germany sample and that from Ghana. Nevertheless, no significant differences were found between Alagoas and Rio de Janeiro, as well as between these two populations and Germany. The combined power of discrimination in males and females were high in both Brazilian populations (≥0.9996 and ≥0.9999998, respectively), showing the utility of these markers for human identification and paternity testing.

Polymorphism and forensic applications of miniSTR loci &lt;I&gt;D10S1248, D2S441, D1S1677, D9S1122&lt;/I&gt;, and &lt;I&gt;D10S1435&lt;/I&gt; in Northwestern Han population

To investigate the polymorphism, genetic stability and the value for analysis degraded samples of D10S1248/ D2S441/D1S1677/D9S1122/D10S1435 five loci in northwestern Han population. The lengths of fragments were analyzed by fluorescence PCR and ABI310 Genetic Analyzer. Samples from 154 unrelated northwestern Han individuals, 10 genealogies and 10 highly degraded specimens were genotyped. Among the 154 unrelated northwestern Han individuals, we discovered 8, 7, 7, 6, and 7 alleles in the five miniSTR loci, respectively. The frequency distributions in the loci showed no deviations from Hardy-Weinberg equilibrium expectations. The H is at 0.662-0.792, PD is at 0.869-0.915, PE is at 0.382-0.585, and PIC is at 0.650-0.750.The research of genetic stability and the value for analysis degraded sample indicated that the five miniSTR loci had high genetic stability and could analysis the degraded samples effectively. Therefore, these 5 miniSTR loci can be used as genetic markers of Northwestern Han populations in forensic practice involving individual identification and paternity testing from degraded DNA samples.

Isolation and characterization of 11 tetranucleotide microsatellite loci in the Egyptian mongoose (Herpestes ichneumon)

We report the isolation of 11 polymorphic tetranucleotide microsatellite loci in the Egyptian mongoose (Herpestes ichneumon). In a sample of 27 individuals, we observed between 4 and 7 alleles per locus and their observed and expected heterozygosities ranged from 0.37 to 0.85 and from 0.44 to 0.79, respectively. All genotypic frequencies conformed to Hardy-Weinberg equilibrium expectations and there were no instances of linkage disequilibrium detected between pairs of loci.

Allele frequencies of 15 STRs in a representative sample of the Brazilian population

Allele frequencies for 15 short tandem repeat (STR) loci were obtained from a sample of 12,030 individuals undergoing paternity testing. This sample includes individuals from all States in Brazil, combined according to the current country division into five regions (North, Northeast, Central West, Southeast, and South). The most polymorphic loci were D2S1338 and D18S51. All the analysed loci meet Hardy–Weinberg equilibrium expectations. Combined power of discrimination and combined power of exclusion for the 15 tested STR loci were 0.999999999999990 and 0.9999992, respectively. Comparative analysis between populations from different Brazilian macroregions as well as between Brazil and other relevant populations are presented.

Microsatellite markers for Pseudoeurycea leprosa, a plethodontid salamander endemic to the Transmexican Neovolcanic Belt

We isolated and characterized 11 polymorphic microsatellite markers for the plethodontid salamander Pseudoeurycea leprosa to obtain population genetic data necessary for the proper management of this threatened species endemic to Central Mexico. We tested polymorphism of these loci among 50 individuals from two populations (Texcalyacac and Calpan) in the states of Mexico and Puebla, across the Transmexican Neovolcanic Belt. The number of alleles per locus ranged from three to 33 (mean; Na = 14.5). Observed and expected heterozygosities ranged from 0.20 to 0.88 and 0.22 to 0.93, respectively. We found deviations from Hardy–Weinberg equilibrium expectations for both populations at two loci (Plt028 and Plt066) and for Calpan population at locus Plt009. Evidence of significant linkage disequilibrium between pairs of loci was found only in one population. We found no evidence of large allele dropouts or stuttering, although null alleles could be present at loci Plt028 and Plt066, which showed significant excess of homozygotes. These markers will be useful for resolving fine-scale population genetic structure, gene flow, and population genetic diversity in Pseudoeurycea leprosa.

Isolation and characterization of microsatellite loci for alligator gar (Atractosteus spatula) and their variability in two other species (Lepisosteus oculatus and L. osseus) of Lepisosteidae

We report on the isolation of 17 polymorphic microsatellite loci from alligator gar (Atractosteus spatula), a large-bodied species that has experienced population declines across much of its range. These loci possessed 2-19 alleles and observed heterozygosities of 0-0.974. All loci conformed to Hardy-Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Nine and eight of these loci were found to be polymorphic in the related species Lepisosteus oculatus and L. osseus, respectively. These microsatellite loci should prove useful in conservation efforts of A. spatula through the study of population structure and hatchery broodstock management.

Low genetic differentiation across three major ocean populations of the whale shark, Rhincodon typus.

BackgroundWhale sharks are a declining species for which little biological data is available. While these animals are protected in many parts of their range, they are fished legally and illegally in some countries. Baseline biological and ecological data are needed to allow the formulation of an effective conservation plan for whale sharks. It is not known, for example, whether the whale shark is represented by a single worldwide panmictic population or by numerous, reproductively isolated populations. Genetic analysis of population structure is one essential component of the baseline data required for whale shark conservation.Methodology/Principal FindingsWe have identified 8 polymorphic microsatellites in the whale shark and used these markers to assess genetic variation and population structure in a panel of whale sharks covering a broad geographic region. This is the first record of microsatellite loci in the whale shark, which displayed an average of 9 alleles per locus and mean Ho = 0.66 and He = 0.69. All but one of the eight loci meet the expectations of Hardy-Weinberg equilibrium. Analysis of these loci in whale sharks representing three major portions of their range, the Pacific (P), Caribbean (C), and Indian (I) Oceans, determined that there is little population differentiation between animals sampled in different geographic regions, indicating historical gene flow between populations. FST values for inter-ocean comparisons were low (P×C = 0.0387, C×I = 0.0296 and P×I = −0.0022), and only C×I approached statistical significance (p = 0.0495).Conclusions/SignificanceWe have shown only low levels of genetic differentiation between geographically distinct whale shark populations. Existing satellite tracking data have revealed both regional and long-range migration of whale sharks throughout their range, which supports the finding of gene flow between populations. Whale sharks traverse geographic and political boundaries during their life history and interbreed with animals from distant populations; conservation efforts must therefore target international protection for this species.

Isolation and characterization of polymorphic microsatellite markers in the grasshopper Mioscirtus wagneri (Orthoptera: Acrididae)

Eight polymorphic microsatellite markers were developed for the grasshopper Mioscirtus wagneri. Polymorphism at these loci was evaluated in 25 individuals from Central Spain. The number of alleles per locus ranged from 3 to 9 and their observed and expected heterozygosities ranged from 0.28 to 0.80 and 0.25 to 0.84, respectively. All genotypic frequencies conformed to Hardy-Weinberg equilibrium expectations, with no evidence of genotypic linkage disequilibrium between any pair of loci. These loci will be highly useful for the study of the population genetic structure and diversity of this grasshopper species forming highly fragmented populations of great conservation concern.

Isolation and characterisation of 11 tetranucleotide microsatellite loci in the common genet (Genetta genetta)

We report the isolation and characterization of 11 polymorphic tetranucleotide microsatellite loci in the common genet (Genetta genetta) from genomic libraries enriched for (AAAG)n and (AGAT)n repeat sequences. We chose to develop tetranucleotide repeats because they can be scored less ambiguously. In a sample of 25 individuals, we observed between four and thirteen alleles per locus and their observed and expected heterozygosities ranged from 0.60 to 0.84 and from 0.68 to 0.92, respectively. All genotypic frequencies conformed to Hardy–Weinberg equilibrium expectations and there were no instances of linkage disequilibrium detected between pairs of loci. These loci will be of use in studies of population genetics, historical demography, and molecular ecology of the common genet.

Microsatellite loci for Ponto‐Caspian gobies: markers for assessing exotic invasions

We developed and tested eight polymorphic microsatellite loci for Ponto-Caspian 'neogobiin' gobies, many of which are invasive in Eurasia and North America, whose study will aid understanding of the population genetics underlying their success. We tested samples from one to two locations from 12 taxa in the recently revised genera Babka, Benthophilus, Mesogobius, Neogobius=Apollonia, Ponticola and Proterorhinus; including the bighead, Caspian, knout, monkey, racer, round, tadpole and tubenose gobies; and taxa from introduced vs. native populations, those diverging between fresh and marine waters, and those differentiated between the Black and Caspian Seas. Populations conformed to Hardy-Weinberg equilibrium expectations, averaging five to 15 alleles per locus and 0.11 to 0.67 mean heterozygosity. Allelic variation significantly differentiated among all taxa and populations.

Population genetics of six miniSTR loci (D1S1677, D2S441, D4S2364, D10S1248, D14S1434, D22S1045) in a Polish population

Allele frequency data and forensic efficiency parameters for six miniSTR loci: D1S1677, D2S441, D4S2364 (NC01), D10S1248, D14S1434, D22S1045 (NC02) were estimated from a sample of 116 unrelated individuals from Poland. No significant deviations from Hardy–Weinberg equilibrium expectations were detected. The combined power of discrimination for the six studied loci was 0.999995383.

Development of polymorphic microsatellite loci for Iranian river buffalo (Bubalus bubalis )

Microsatellite loci were developed using PCR-based isolation of microsatellite arrays (PIMA) for Iranian river buffalo. Blood samples of eighty unrelated individuals from four buffalo populations (Khuzestan, Mazandaran, Guilan and Azarbayejan) were taken and following DNA extraction, isolation of microsatellite loci initiated using enrichment with random amplified polymorphic DNA (RAPD) primers. RAPD-PCR fragments were ligated into PTZ57R TA cloning vector and transformed into DH5
competent cells. Obtained colonies were screened for presence of repetitive elements by repeatspecific and M13 forward and reverse primers. After designing primer pairs for repeat containing fragments, they were tested in all buffalo populations. Two microsatellite loci (RBBSI and RBBSII) were informative and polymorphic. Number of alleles for RBBSI and RBBSII in 80 individuals was 5 and 6, respectively. Expected heterozygosity ranged from 0.65 to 0.81. Significant deviation from Hardy-Weinberg equilibrium expectation occurred for both loci in all populations, but 37.5% of locus/population combination showed the deviation. We postulate that the two newly isolated microsatellite loci during this study could be useful for population genetic studies in Bubalus bubalis.