Variability of microsatellite markers in Helicopsis (Gastropoda; Pulmonata) steppe mollusks in the southern Central Russian upland

Genetic variation of Anopheles gambiae was analysed to assess interpopulation divergence over a 6000 km distance using short tandem repeat (microsatellite) loci and allozyme loci. Differentiation of populations from Kenya and Senegal measured by allele length variation at five microsatellite loci was compared with estimates calculated from published data on six allozyme loci (Miles, 1978). The average Wright's FST of microsatellite loci (0.016) was lower than that of allozymes (0.036). Slatkin's RST values for microsatellite loci were generally higher than their FST values, but the average RST value was virtually identical (0.036) to the average allozyme FST. These low estimates of differentiation correspond to an effective migration index (Nm) larger than 3, suggesting that gene flow across the continent is only weakly restricted. Polymorphism of microsatellite loci was significantly higher than that of allozymes, probably because the former experience considerably higher mutation rates. That microsatellite loci did not measure greater interpopulation divergence than allozyme loci suggested constraints on microsatellite evolution. Alternatively, extensive mosquito dispersal, aided by human transportation during the last century, better explains the low differentiation and the similarity of estimates derived from both types of genetic markers.

The relationship between heterozygosity at genetic markers (six allozyme and eight microsatellite loci), and fluctuating asymmetry (FA), length and weight was investigated in two samples of Atlantic salmon (Salmo salar L.) with different timings of first active feeding (early (EA) and late (LA) salmon). This trait had previously been related to fitness. EA fish show smaller values of FA, are longer, heavier and are more heterozygous at allozyme loci than are conspecific LA fish. Also within both samples, heterozygosity at allozyme loci was inversely related to FA and was positively related to weight and length. However, no significant differences in microsatellite diversity (heterozygosity and mean d2 measurements) were observed between samples (EA vs LA). Furthermore, no association was observed between the variability at microsatellite loci and FA, weight or length within each sample. These results suggest that allozyme loci, in themselves, influence fitness components, rather than associations arising from associative overdominance.

<p>New Zealand has a long history of isolation and has evolved a unique biota. Spanning from a sub-tropical climate in the North to a sub-Antarctic climate in the far south New Zealand provides an interesting opportunity to study the processes that lead to evolution. This thesis attempts to study the evolution of Ostrea chilensis at a population genetics and molecular level. Chapter Two: Microsatellite DNA loci represent an ideal marker for population genetics studies due to high levels of length polymorphism between individuals. Genomic sequencing technologies offer the potential to quickly identify thousands of loci, from which PCR primers can be developed and screened for polymorphisms. I aimed to develop PCR primers to amplify length polymorphic microsatellite loci and to use the genomic data set to elucidate patterns and processes of microsatellite evolution. DNA was extracted from a single Ostrea chilensis individual and used for a 1/8 plate sequencing run on a Roche 454. The subsequent quality checked DNA database was annotated for microsatellite loci. 6,208 dinucleotide, 7,326 trinucleotide repeats, 2,414 tetranucleotide, 33 hexanucleotide and 356 pentanucleotides were annotated on the partial genome. Four microsatellite loci were successfully amplified and genotyped. The loci have a low number of alleles compared to other bivalve studies and two have significant departures from HWE (Fst = 0.126 and -0.348). There were a number of highly significant BLAST hits (< 1xE⁻²⁰) with repetitive Oyster DNA sequences obtained from GenBank. Due to difficulties the microsatellite loci were abandoned as markers for later population genetic analysis. This work, however, provides the ground work for further developments of PCR primers for polymorphic microsatellite DNA and provides some observations of molecular evolution of repetitive DNA, which will lead to a greater understanding of these sequences. Chapter Three: This chapter forms the first population genetic study of Ostrea chilensis using New Zealand and Chilean populations. The life history traits of O. chilensis are thought to reduce the dispersal of the species. Using randomly amplified polymorphic DNA (RAPD) I aimed to test the population genetic structure with the null hypothesis that there is panmixia (i.e. high levels of gene flow and no barriers between populations). I then aimed to see if there was an isolation-by-distance (IBD) profile. Barriers to gene flow at around 420S have been identified in a number of studies around New Zealand. I aimed to see if those barriers were present in this study. Significant spatial genetic differentiation was found among populations (Fst= 0.194, p<0.00001). Over all spatial scales a significant IBD was not found, until ‘outlier’ (those with two standard deviations from the mean Fst) were removed, then a slight IBE was found (rxy=0.324, p=0.030, r2 =0.1052). In an AWclust analysis two main clusters were revealed, but they did not correspond to above and below the 42⁰S. It is possible that the brooding life style of Ostrea chilensis has resulted in reduced gene flow between populations. Much of the genetic structure was not congruent with geographic location; this apparent chaotic patchiness could be influenced by human mediated movements and/or environmental variables. Chapter Four: The genetic structure found in the previous chapter was analysed in the context of near shore environmental and geo-spatial variables. My aim was to elucidate the environmental variables that best explain the apparent genetic structure of O. chilensis. Using genetic data from the previous chapter a Generalised Linear Model (GLM) was used to test the effect of ten environmental variables and two geospatial variables on average genetic distance (Fst and PHIst). A complimentary BEST analysis was used to test the effect of the same variables on the individual alleles for each population. Using the BEST analysis the model that best explained the apparent genetic structure (Rs = 0.263) included the environmental variables Sediment (SED) and mean sea surface temperature (SSTGrad). The GLM showed a more complicated model including most of the variables tested. Previous studies have shown that bivalve dispersal patterns are associated with sediment and spawning times can be influenced by temperature. This line of enquiry is important as it could lead to the identification of candidate genes for selection. Chapter Five: Genomic datasets contain a wealth of data that can be used to further understand genome structure and arrangements. My aim was to discover mitochondrial gene fragments in the genomic data set, use them to assemble the Ostrea chilensis mitogenome, and then analyse the genome in a phylogenetic framework with other oyster mitogenomes. A custom BLAST database was created using the 454 genomic DNA data set; this was then BLASTED against all available oyster mitogenomes on GenBank. The DNA sequences with good statistical support (<1 x E⁻²⁰) where mapped against the mitogenome of Ostrea edulis. The O. edulis mitogenome was then used to annotate the resulting mitogenome. I was able to recover 10, 086 bp of mitochondrial DNA – this represents around 65% of the full genome. The subsequent topology of the Bayesian phylogenetic tree was similar to that found in previous studies. PCR primers have been designed to sequence the gaps in the mitogenome. This will allow full annotation. Full genome annotation will aid further research into genome evolution. Chapter Six: In the mitogenome, evolution is thought to mainly result in synonymous substitutions, due to functional constraints. I aimed to describe the selection pressures acting on each protein coding gene of the oyster mitogenome by comparing rates of non-synonymous (dN) and synonymous (dS) substitutions. I then aimed to discover if there was a codon bias in the oyster mitogenomes. Genetic distance was assessed using p-distance. The ratio of dN/dS was calculated using the method of Nei and Gojobori (1986). The overall ratio of dN/dS was <1 for all protein coding genes, this would suggest that the genes are under purifying selection (non-synonymous mutations are selected against). A consistent codon bias was found across all protein coding genes, this could indicate translational selection. This multidisciplinary approach aimed to explain the patterns and processes of evolution in O. chilensis. This thesis research developed molecular tools, and provided information that will aid fisheries and aquaculture management.</p>

The freshwater snail Biomphalaria glabrata is the principal intermediate host for the parasite Schistosoma mansoni within Brazil. We assessed the potential effects of snail population dynamics on parasite transmission dynamics via population genetics. We sampled snail populations located within the confines of three schistosome-endemic villages in the state of Minas Gerais, Brazil. Snails were collected from individual microhabitats following seasonal periods of flood and drought over the span of 1year. Snail spatio-temporal genetic diversity and population differentiation of 598 snails from 12 sites were assessed at seven microsatellite loci. Average genetic diversity was relatively low, ranging from 4.29 to 9.43 alleles per locus, and overall, subpopulations tended to exhibit heterozygote deficits. Genetic diversity was highly spatially partitioned among subpopulations, while virtually, no partitioning was observed across temporal sampling. Comparison with previously published parasite genetic diversity data indicated that S.mansoni populations are significantly more variable and less subdivided than those of the B.glabrata intermediate hosts. Within individual Brazilian villages, observed distributions of snail genetic diversity indicate temporal stability and very restricted gene flow. This is contrary to observations of schistosome genetic diversity over the same spatial scale, corroborating the expectation that parasite gene flow at the level of individual villages is likely driven by vertebrate host movement.

Genetic variation at 19 allozyme (including 11 polymorphic) and 10 microsatellite loci was examined in the population samples of odd- and even-broodline pink salmon from the southern part of Sakhalin Island, Southern Kuril Islands, and the northern coast of the Sea of Okhotsk. The estimates of relative interpopulation component of genetic variation over the allozyme loci, per broodline, were on average 0.43% (GST), while over the microsatellite loci it was 0.26% (the theta(ST) coefficient, F-statistics based on the allele frequency variance), and 0.90% (the rho(ST) coefficient, R-statistics based on the allele size variance). The values of interlinear component constituted 2.34, 0.31, and 1.05% of the total variation, respectively. Using the allozyme loci, statistically significant intralinear heterogeneity was demonstrated among the regions, as well as among the populations of Southern Sakhalin Island. Multivariate scaling based on the allozyme data demonstrated regional clustering of the sample groups, representing certain populations during the spawning run or in different years. Most of the microsatellite loci examined were found to be highly polymorphic (mean heterozygosity > 0.880). The estimates of interlinear, interregional, and interpopulation variation over these loci in terms of theta(ST) values were substantially lower than in terms of rho(ST) values. Regional genetic differentiation, mostly expressed at the allozyme loci among the populations from the northern and southern parts of the Sea of Okhotsk (i.e., between the Sakhalin and Kuril populations), was less expressed at the microsatellite loci. The differentiation between these regions observed can be considered as the evidence in favor of a large-scale isolation by distance characterizing Asian pink salmon. It is suggested that in pink salmon, low genetic differentiation at neutral microsatellite loci can be explained by extremely high heterozygosity,of the loci themselves, as well as by the migration gene exchange among the populations (the estimate of the genetic migration coefficient inferred from the "private" allele data constituted 2.6 to 3.4%), specifically, by the ancient migration exchange, which occurred during postglacial colonization and colonization of the range.

White Sands pupfish (Cyprinodon tularosa) are endemic to southern New Mexico and occur in only four localities: Malpais Spring, Salt Creek, Mound Spring and Lost River. Recently reported historical accounts indicate that the latter two populations were derived from translocations. Their limited distribution and complicated history suggest that knowledge of population genetic structure would be useful for the development of a sound conservation strategy. Mitochondrial DNA (mtDNA) sequences for a segment of the control region showed little variation. Variation was observed for microsatellite and allozyme loci with 37% attributable to divergence among populations. The mean genetic distance between Malpais Spring and the other three populations was high (allozymes: Darc= 0.541; microsatellites: Rst= 0.684) compared with the mean distance among the other three populations (Darc=0.161, Rst=−0.016). There were fixed or nearly fixed differences in allele frequency between the Malpais Spring population and the other three populations at one allozyme locus (hexokinase) and two microsatellite loci (WSP‐02 and WSP‐11). We suggest the recognition of two evolutionarily significant units (ESUs) for the White Sands pupfish: Malpais Spring and Salt Creek. Our data indicate that the Lost River and Mound Spring populations descended from translocations from the Salt Creek population. Therefore, conservation efforts should focus on the Malpais Spring population.

Historical metal pollution in natural gudgeon populations: Inferences from allozyme, microsatellite and condition factor analysis

BackgroundThe elimination of schistosomiasis remains a challenging task, with current measures primarily focused on the monitoring and control of Oncomelania hupensis (O. hupensis) snail, the sole intermediate host of Schistosome japonicum. Given the emerging, re-emerging, and persistent habitats of snails, understanding their genetic diversity might be essential for their successful monitoring and control. The aims of this study were to analyze the genetic diversity of Oncomelania hupensis robertsoni (O. h. robertsoni) using microsatellite DNA markers; and validate the applicability of previously identified microsatellite loci for O. hupensis in hilly regions.MethodsA total of 17 populations of O. h. robertsoni from Yunnan Province in China were selected for analysis of genetic diversity using six microsatellite DNA polymorphic loci (P82, P84, T4-22, T5-11, T5-13, and T6-27).ResultsThe number of alleles among populations ranged from 0 to 19, with an average of 5. The average ranges of expected (He) and observed (Ho) heterozygosity within populations were 0.506 to 0.761 and 0.443 to 0.792, respectively. The average fixation index within the population ranged from – 0.801 to 0.211. The average polymorphic information content (PIC) within the population ranged from 0.411 to 0.757, appearing to be polymorphic for all loci (all PIC > 0.5), except for P28 and P48. A total of 68 loci showed significant deviations from Hardy-Weinberg equilibrium (P < 0.05), and pairwise Fst values ranged from 0.051 to 0.379. The analysis of molecular variance indicated that 88% of the variation occurred within snail populations, whereas 12% occurred among snail populations. Phylogenetic trees and principal coordinate analysis revealed two distinct clusters within the snail population, corresponding to “Yunnan North” and “Yunnan South”.ConclusionsO. h. robertsoni exhibited a relatively high level of genetic differentiation, with variation chiefly existing within snail populations. All snail in this region could be separated into two clusters. The microsatellite loci P82 and P84 might not be suitable for classification studies of O. hupensis in hilly regions. These findings provided important information for the monitoring and control of snail, and for further genetic diversity studies on snail populations.Graphical

The artificial movement of individuals between populations (translocation) can be an effective way to increase genetic diversity within populations, but few studies have undertaken long term genetic monitoring to determine if variation introduced by translocation is maintained over many generations or whether it can be used to adapt to local conditions. Here, we report on the changes in morphological and molecular variation over a 12-year period in a population of an intertidal littorine snail (Bembicium vittatum) that was created by mixing individuals from three geographically disjunct populations. These source populations differ genetically in shell shape and in allele frequency at several allozyme loci. We found that the translocated population had higher allozyme diversity than any of the source populations and that this pattern was maintained over multiple generations. Variation in shell shape also increased, but this declined over time as shells became taller. Some allozyme loci also showed significant changes in frequency over time. These changes were not consistently towards the genetic makeup of a single source population, and in the case of shell shape, were towards a phenotype that was most suited to the local environment. Our results suggest that genetic variation introduced into a population by translocation can be rapidly incorporated and used to adapt to local conditions without domination by a single source population’s genome. However, more studies are needed before generalisations on the benefits of mixing individuals from disjunct populations can be made.

Relationships of two species of Channa argus (northern snakeheads) named “Bicolor type” and “White type”, as well as other six Channa species were investigated based on their partial sequence of mitochondrial DNA 16S rRNA genes in the present study. For the Channa family, the average genetic distance was 0.0863 with the inter-species genetic distance ranged from 0.0173 to 0.1384, and the average intra-species genetic distance in the genus channa was estimated as 0.00273 (range: 0.0019 to 0.0038). For the two C. argus species, the mean pair-wise genetic distance between “Bicolor type” and “White type” northern snakehead was estimated as 0.00218, which was within the intra-species genetic distance interval for Channa species, indicating that they belonged to the same species at molecular level. Moreover, these snakeheads can be divided into two distinct groups via 16S rRNA, which might be more accurate for Channa classification than traditional method based on the absence or presence of the pelvic fins of the fish.

Longtail Tuna (Thunnus tonggol) is one of the neritic species of tuna from the Scrombridae family. T. tonggol is oceanicromus and ussually found in tropical and subtropical waters in the Indo-Pacific region. Although known as one of the tuna species, the information of this species is very lacking, especially in Indonesia. Therefore, this study aims to determine the genetic diversity and kinship relationships of longtail tuna (T. tonggol) collected at Sagulung fish market, Batam, Riau Islands. Molecular analysis were carried out using genetic markers namely mitochondrial D-loop (control regian) with the stages of DNA extraction, PCR (Polymerase Chain Reaction), electrophoresis, sequencing, and data analysis. A total of 21 longtail tuna samples were identified using molecularly and confirmed as T. tonggol species, which have a base length ranging from 482 - 523 bp (base pairs). The value of haplotype diversity (Hd) of longtail tuna was 1,00000 and the value of nucleotide diversity (?) was 0,01654. The results of phylogenetic tree reconstruction showed that all samples collected were in the same group (clade) with the average genetic distance in one T. tonggol clade obtained a value of 0.010 which has a close relationship between samples. This result can be use as additional information for longtail tuna species in Indonesia.

Genetic variability and structure of common carp ( Cyprinus carpio) populations throughout the distribution range inferred from allozyme, microsatellite and mitochondrial DNA markers

We compared patterns of genetic structure at potentially selected (two allozyme loci) and neutral molecular markers (six microsatellite loci) in the acorn barnacle, Semibalanus balanoides from the Gulf of St. Lawrence. Our results confirmed the presence of a geographical shift in alleles MPI and GPI near the Miramichi River. In contrast, no significant patterns of population differentiation among samples located north and south of the river mouth were detected for four of six microsatellite loci. However, analysis of molecular variance (amova) at individual loci revealed that a significant proportion of the total variance in allele frequencies was partitioned among samples located north and south of the river for both the allozyme and the other two microsatellite loci. The two most common alleles at these microsatellites showed frequencies that were highly correlated (r = 0.65-0.74, P < 0.05) with those of the MPI*2 allele, perhaps because of either physical linkage or epistasis. The two allozyme loci were significantly correlated in barnacles located north of the Miramichi River (r = 0.86, P < 0.05). Overall, our results supported the hypothesis that the broad scale pattern of allozyme allelic shifts is maintained by selection. They also indicated that microsatellites may not always behave in a neutral way and must be used cautiously, especially when evidence for genetic structuring relies on only a few assayed loci.

Natural selection has been invoked to explain the observed geographic distribution of allozyme allele frequencies for a number of teleost species. The effects of selection on allozyme loci in three species of Pacific salmon were tested. A simulation-based approach to estimate the null distribution of population differentiation (F ST) and test for F ST outliers was used. This approach showed that a majority of allozyme loci conform to neutral expectations predicted by the simulation model, with relatively few F ST outliers found. No consistent F ST outlier loci were found across species. Analysis of population sub-groups based on geography and genetic identity reduced the number of outlier loci for some species, indicating that large geographic groups may include genetically divergent populations and/or that there is geographic heterogeneity in selection pressure upon allozyme loci. Two outlier allozyme loci found in this analysis, lactate dehydrogenase-B and malic enzyme, have been shown to be influenced by selection in other teleost species. This approach is also useful in identifying allozyme loci (or other genetic markers) that meet assumptions for population genetic study.

This paper reports data on 28 allozyme loci in wild and artificially reared sea bass (Dicentrarchus labrax) samples, originating from either coastal lagoon or marine sites in the Mediterranean Sea. F ST analysis (θ estimator) indicated strong genetic structuring among populations; around 34% of the overall genetic variation is due to interpopulation variation. Pairwise θ estimates showed that, on average, the degree of genetic structuring was much higher between marine populations than between samples from lagoons. Six polymorphic loci showed differences in allele frequencies between marine and lagoon samples. Multivariate analyses of individual allozymic profiles and of allele frequencies suggested that different arrays of genotypes prevail in lagoons compared to marine samples, particularly at those loci that, on the basis of previous acclimation experiments, had been implicated in adaptation to freshwater. On the other hand, variation at “neutral” allozyme loci reflects to a greater extent the geographic location of populations. Allozyme differentiation was also studied in a D. labrax population from the Portuguese coast. Average genetic distance between this population and the Mediterranean populations was quite high (Nei's D = 0.236) and calls into question the taxonomic status of the Portuguese population. Finally, genetic relationships between D. labrax and D. punctatus were evaluated. Average Nei's D was 0.648, revealing high genetic differentiation between the two species, even for two sympatric populations of these species in Egypt; thus gene flow was not indicated between species.