Microsatellite loci to assess genetic variation in Tor putitora

Seven polymorphic microsatellite DNA loci were identified in golden mahseer, Tor putitora, through cross‐species amplification. Thirty‐two primers developed for three cyprinid fishes were tested in the study. The genetic variation detected at each microsatellite locus in T. putitora specimens (n = 107), collected from three different rivers and one lake was assessed. The allele frequencies deviated significantly from that expected under Hardy–Weinberg equilibrium. The mean observed heterozygosity values ranged from 0.29 to 0.40. Significant genotype heterogeneity indicated that the samples were not drawn from the same gene pool. The results indicate that the identified microsatellite loci exhibit promise for use in fine scale population structure analyses of T. putitora.     

Microsatellite markers help to assess genetic diversity among Opuntia ficus indica cultivated genotypes and their relation with related species

Opuntia spp. belong to the Cactaceae family and are native to Central America. The most economically important species is O. ficus indica, cultivated both for fruits and cladodes. The genus includes other important edible species (from diploid to octoploid) that occur worldwide as either wild or cultivated species in many arid or semiarid areas (e.g., the Mediterranean region). Several accessions are cultivated in different growing regions, but little is known about their ancestries and levels of genetic diversity. The aim of this study was to investigate the level of intraspecific genetic diversity among O. ficus indica cultivated varieties and some related species. Specifically, six highly polymorphic simple sequence repeats (SSR) and two expressed sequence tag (EST)-SSR loci were investigated in 62 wild and cultivated genotypes belonging to 16 Opuntia species. The clusters identified by the distance and model-based analyses clearly separated the wild opuntias from the cultivated ones. However, the O. ficus indica accessions did not cluster separately from other arborescent cactus pear species, such as O. amyclaea, O. megacantha, O. streptacantha, O. fusicaulis, and O. albicarpa, indicating that their current taxonomical classifications do not fit with their genetic variability. In general, the genotypes cultivated in Mexico showed high levels of diversity, whereas most of the spineless accessions collected in other countries had a very narrow genetic base. This study increases our knowledge of the variability among some of the most diffused Opuntia cultivated accessions. This study also points to the inconsistencies of previous taxonomical genotype assignments that were based solely on morphological characteristics.     

Use of a multivariate model using allele frequency distributions to analyse patterns of genetic differentiation among populations

Very few studies have attempted to relate the properties of some ordination techniques to classical tools of population genetics as F -statistics. A multivariate model to analyse population genetics data based on the properties of 'joint scaling' of populations and loci is developed. The design of population genetics data means that this model deals with a modified version of the classical Multiple Correspondence Analysis which is called Constant Row Total-Multiple Correspondence Analysis (CRT-MCA) and is an original tool in population genetics. Such a model allows estimates of the degree of population differentiation by studying the variability of the distribution of allele frequencies in different samples. Some clear relationships exist between some model parameters and the classical F_st statistics. The CRT-MCA also allows all the studied loci to be considered simultaneously and the role of each locus in patterns of population differentiation to be expressed. Such a multivariate approach prevents the use of any pooling strategy as is classically used in studies of hierarchical F -statistics. The relevance of the CRT-MCA model is illustrated by the analysis of population structure of 15 dogwhelk ( Nucella lapillus ) populations in south-west England. The advantages and limitations of CRT-MCA are presented.     

Microsatellite allele ladders in two species of Pacific salmon: preparation and field‐test results

To support microsatellite data communication, we have developed a convenient method for creating locus‐specific microsatellite allele ladders used to align data from different laboratories. The ladders were constructed by pooling polymerase chain reaction (PCR) products to create a template for amplification. Four ladders were field‐tested in six different laboratories using different genotyping platforms. Despite substantial differences in absolute size estimates of DNA fragments, each laboratory correctly scored unknown sample genotypes according to the ladder designations. The results indicate that our simple preparation method provides reliable allele ladders in a time‐efficient manner for verifying microsatellite genotypes across platforms.     

Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting

Many of the Haemonchus contortus isolates currently used for experimental work were originally derived from different regions of the world and are commonly exchanged between laboratories. In most cases, these are largely genetically uncharacterised other than the analyses conducted on specific genes of interest. We have used a panel of eight microsatellite markers to genetically characterise five different commonly used H. contortus isolates including MHco3 (ISE), the isolate chosen for full genome sequencing as part of the H. contortus genome project. There is an extremely high level of genetic differentiation between each of the isolates except the two which have a common origin, MHco1 (MOSI) and MHco3 (ISE). We have investigated the amplification of microsatellite markers from pooled DNA as a potential method for fingerprinting different isolates. Good estimates of the true allele frequencies can be made by amplification from either pooled adult DNA or bulk L3 DNA for seven out of the eight markers tested. Both single worm genotyping and bulk DNA fingerprinting revealed no genetic differentiation between adult worms in the host and larvae derived from faecal culture. Furthermore, none of the eight markers showed genetic changes when isolates were passaged through different individual hosts. Hence the microsatellite genotyping of bulk larval DNA samples provides a simple and rapid method to genetically define and monitor laboratory isolates, and to determine their relationship with particular field populations.     

A simple method of removing the effect of a bottleneck and unequal population sizes on pairwise genetic distances

In this paper, we derive the expectation of two popular genetic distances under a model of pure population fission allowing for unequal population sizes. Under the model, we show that conventional genetic distances are not proportional to the divergence time and generally overestimate it due to unequal genetic drift and to a bottleneck effect at the divergence time. This bias cannot be totally removed even if the present population sizes are known. Instead, we present a method to estimate the divergence times between populations which is based on the average number of nucleotide differences within and between populations. The method simultaneously estimates the divergence time, the ancestral population size and the relative sizes of the derived populations. A simulation study revealed that this method is essentially unbiased and that it leads to better estimates than traditional approaches for a very wide range of parameter values. Simulations also indicated that moderate population growth after divergence has little effect on the estimates of all three estimated parameters. An application of our method to a comparison of humans and chimpanzee mitochondrial DNA diversity revealed that common chimpanzees have a significantly larger female population size than humans.     

Exploiting a wheat EST database to assess genetic diversity

Expressed sequence tag (EST) markers have been used to assess variety and genetic diversity in wheat (Triticum aestivum). In this study, 1549 ESTs from wheat infested with yellow rust were used to examine the genetic diversity of six susceptible and resistant wheat cultivars. The aim of using these cultivars was to improve the competitiveness of public wheat breeding programs through the intensive use of modern, particularly marker-assisted, selection technologies. The F₂ individuals derived from cultivar crosses were screened for resistance to yellow rust at the seedling stage in greenhouses and adult stage in the field to identify DNA markers genetically linked to resistance. Five hundred and sixty ESTs were assembled into 136 contigs and 989 singletons. BlastX search results showed that 39 (29%) contigs and 96 (10%) singletons were homologous to wheat genes. The database-matched contigs and singletons were assigned to eight functional groups related to protein synthesis, photosynthesis, metabolism and energy, stress proteins, transporter proteins, protein breakdown and recycling, cell growth and division and reactive oxygen scavengers. PCR analyses with primers based on the contigs and singletons showed that the most polymorphic functional categories were photosynthesis (contigs) and metabolism and energy (singletons). EST analysis revealed considerable genetic variability among the Turkish wheat cultivars resistant and susceptible to yellow rust disease and allowed calculation of the mean genetic distance between cultivars, with the greatest similarity (0.725) being between Harmankaya99 and Sönmez2001, and the lowest (0.622) between Aytin98 and Izgi01.     

Bilateral phrenic stimulation: a simple technique to assess diaphragmatic fatigue in humans

Transdiaphragmatic pressure (Pdi) and the rate of relaxation of the diaphragm (tau) were measured at functional residual capacity (FRC) in six normal seated subjects during single-twitch stimulation of both phrenic nerves. The latter were stimulated supramaximally with needle electrodes with square-wave impulses of 0.1-ms duration at 1 Hz before and after diaphragmatic fatigue produced by resistive loaded breathing. Constancy of chest wall configuration was achieved by monitoring the diameter of the abdomen and the rib cage with a respiratory inductive plethysmograph system. During control the peak Pdi generated during the phrenic stimulation amounted to 34.4 +/- 4.2 (SE) cmH2O and represented in each subject a fixed fraction (17%) of its maximal transdiaphragmatic pressure. After diaphragmatic fatigue the peak Pdi decreased by an average of 45%, amounting to 18.1 +/- 2.7 cmH2O 5 min after the fatigue run, and tau increased from 55.2 +/- 9 ms during control to 77 +/- 8 ms 5 min after the fatigue run. The decrease in peak Pdi and the increase in tau observed after the fatigue run persisted throughout the 30 min of the recovery period studied, the peak Pdi amounting to 18.4 +/- 2.8 and 18.9 +/- 3.3 cmH2O and tau to 81.3 +/- 5.7 and 88.7 +/- 10 ms at 15 and 30 min after the end of the fatigue run, respectively. It is concluded that diaphragmatic fatigue can be detected in man by bilateral phrenic stimulation with needle electrodes without any discomfort for the subject and that the decrease in diaphragmatic strength after fatigue is long lasting.     

Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus.

In normal human fibroblasts, the synthesis of a cell surface receptor for plasma low density lipoprotein (LDL) is regulated by a sensitive system of feedback suppression. The number of functional LDL receptors declines by more than 20 fold when cellular stores of esterified cholesterol are increased by incubation of cells with an exogenous source of cholesterol. Fibroblasts from patients with the heterozygous form of familial hypercholesterolemia (FH) possess one functional allele and one nonfunctional allele at the LDL receptor locus. In the current studies, we have examined the effect that this deficiency produces upon the pattern of regulation of the single functional allele at the LDL receptor locus. Under growth conditions that induced a maximal rate of LDL receptor synthesis (that is, growth in the absence of an exogenous source of cholesterol), the FH heterozygote cells produced about one half as many functional LDL receptors as did the normal cells. More importantly, when grown in the presence of increasing amounts of exogenous cholesterol, the FH heterozygote and normal cells suppressed their respective LDL receptor activities in parallel. Over a wide range of LDL receptor activities, at each level of cellular esterified cholesterol, the FH heterozygote cells expressed about one half as many receptors as did the normal cells. These data indicate that in the FH heterozygote cells, the receptor regulatory mechanism dictates that the normal allele produce only the amount of gene product that it would normally produce at a given level of cellular esterified cholesterol. The failure of the regulatory mechanism to stimulate the normal allele at the LDL receptor locus to produce twice its normal amount of gene product leaves the FH heterozygote cells with a persistent 50% deficiency in LDL receptors under all conditions of cell growth.     

Four-cluster analysis: a simple method to test phylogenetic hypotheses

A simple statistical test for comparing three alternative phylogenetichypotheses for four monophyletic groups is presented. This test is based onthe minimum-evolution principle, and it does not require any informationregarding the branching order within each monophyletic group. It iscomputationally efficient and can be easily extended to five or moremonophyletic groups.     

Contributions of expression redistribution and allele substitutions to the genetic differentiation of animal taxa of different ranks

The contributions to the genetic differences between animal taxa of various ranks were estimated for four groups of loci: loci with identical (i), similar (s), or completely different (d) allele compositions, and (o) loci displaying a change in expression. The main contribution is made by i-and s-loci at the intraspecific level. Another pattern is observed at the specific level: d-loci become dominant and o-loci appear, suggesting fundamental changes in the genomes upon speciation. The relationships between the contributions of the four groups of loci are relatively stable within a class and differ among classes. Interestingly, the extent of genetic differentiation (as estimated from the proportions of the above four components) observed at the specific level in amphibians is achieved only at the level of families in birds. The approach is especially efficient for identifying hybrid populations and individuals.     

Microsatellite diversity among three endogamous Tamil populations suggests their origin from a separate Dravidian genetic pool

The genetic profiles based on 15 autosomal microsatellite markers were analyzed among three socially distinct endogamous Dravidian populations: Tanjore Kallar, Vanniyar, and Pallar of Tamil Nadu, southern India, in order to understand their origin and the extent of genetic affinity and diversity among them. All loci were highly polymorphic and followed Hardy-Weinberg expectations except for loci D13S317 in Tanjore Kallars and D7S820 in Vanniyars. The SK2 criterion test showed no evidence of association among the 15 loci in the studied populations. The extent of gene differentiation among the three populations was low (G(ST) = 0.012), suggesting proximity between them. The phylogenetic dendrogram based on allele frequencies places them in a separate cluster, away from other compared Indo-European populations. The fit of the Harpending and Ward model of regression was found to be good and consistent with the extent of endogamy followed by the respective populations. These findings support a separate origin of the Dravidians and reveal an overall genetic unity among the studied Tamil populations belonging to different strata of the social hierarchy. The extent of diversity found among them probably resulted from the strict endogamous practices that they follow.     

The ParaSight-F test: a simple rapid manual dipstick test to detect Plasmodium falciparum infection

A rapid diagnostic for Plasmodium falciparum based on an antigen capture has been incorporated in a simple, easily interpreted dipstick by Becton Dickinson Advanced Diagnostics. In this article Clive Shiff, Japhet Minjas and Zul Premji discuss its evaluation in rural Tanzania and the implications of such a test in handling malaria cases under field conditions.     

Screening of poplar biomass for bio-active compounds: a simple method to assess antioxidant activity

Poplar bud resinoids are a potential source of natural antioxidants. As poplar culture today involves many hybrids, a simple screening test to assess antioxidant properties was proposed. This method used the second derivative of the UV spectra at 233 nm of the iron induced peroxidienes resulting from linoleic acid peroxidation. Kinetic data showed a lag period followed by a quadratic increase in peroxidienes. These two phases were more clearly separated using the square root of the data. An acceptable linear fitting of the length of the lag phase with antioxidant concentration was observed. Calibrating the experimental test with BHA therefore allowed an antioxidant assessment as "BHA equivalent". First results clustered well with taxonomic data, with typically 0.5, 0.15 and 0.08 "BHA equivalent" for P. nigra, P. X euramericana and P. X interamericana, respectively.     

GLOSSI: a method to assess the association of genetic loci-sets with complex diseases

Background  

The developments of high-throughput genotyping technologies, which enable the simultaneous genotyping of hundreds of thousands of single nucleotide polymorphisms (SNP) have the potential to increase the benefits of genetic epidemiology studies. Although the enhanced resolution of these platforms increases the chance of interrogating functional SNPs that are themselves causative or in linkage disequilibrium with causal SNPs, commonly used single SNP-association approaches suffer from serious multiple hypothesis testing problems and provide limited insights into combinations of loci that may contribute to complex diseases. Drawing inspiration from Gene Set Enrichment Analysis developed for gene expression data, we have developed a method, named GLOSSI (Gene-loci Set Analysis), that integrates prior biological knowledge into the statistical analysis of genotyping data to test the association of a group of SNPs (loci-set) with complex disease phenotypes. The most significant loci-sets can be used to formulate hypotheses from a functional viewpoint that can be validated experimentally.     

A simple regression model to assess environmental effects on fish growth

Multiple regression was used to assess relationships between annular growth and environmental variables. This approach (1) tests for the significance of environmental changes on fish growth and (2) can be successfully used to predict fish growth using different environmental conditions. Age usually explains a large proportion of the variation in growth increments measured in length, and these two variables are inversely related. Since length increments decline as age increases, environmental impacts on growth will be reduced as fish grow older. To account for within-age growth variation and incorporate this age-dependent factor, the inverse of age (1/age in years) is multiplied by the value of an environmental variable (ENV) that may be related to growth. This interaction term and age are regressed against mean annular growth increments in length (TLINC; l _{t +1}— l ₁):
TLINC = b₀—b₁AGE±b₂(1/AGE)*ENV
where b₀, b₁, and b₂ are the regression coefficients for intercept and slope, respectively. Additional variables that measure environmental factors can be added to the model. Environmental effects associated with growth rates of black crappie, Pomoxis nigromaculatus (LeSueur), are presented as an example.     

Microsatellite markers to assess the influence of population size, isolation and demographic change on the genetic structure of the UK butterfly Polyommatus bellargus

Five microsatellite DNA markers were isolated and used to quantify population genetic structure among a subset of UK populations of the Adonis blue (Polyommatus bellargus Rottemburg). Specifically, whether population size, degree of isolation or history of bottlenecking in 1976-1978 can explain current patterns of genetic variation. The butterfly is at its northern range limit in the UK, where it exists as a highly fragmented metapopulation on isolated pockets of calcareous grassland. Most populations were affected by a severe bottleneck in the late 1970s, when a drought caused the host plant (Hippocrepis comosa) to wilt. Mantel tests and spatial autocorrelation analysis indicated a significant effect of isolation by distance among the UK populations, a relationship that broke down at greater geographical scales (> 23.85 km), probably because of large areas of unsuitable habitat presenting barriers to gene flow. Similarly, amova revealed that variation among geographical regions was almost double that observed within regions. Larger populations were found to support significantly higher levels of genetic diversity, suggesting that small populations may lose genetic diversity through drift. If, as in other butterfly species, low genetic diversity increases the probability of population extinction, then these populations are likely to be under threat. Neither isolation nor a history of bottlenecks appeared to influence genetic diversity. The results indicate that adequate population size a crucial factor in the conservation of genetic diversity in P. bellargus in the UK.