Microsatellites and kinship

Many evolutionary studies, particularly kinship studies, have been limited by the availability of segregating genetic marker loci. Microsatellites promise to alleviate these problems. Microsatellite loci are segments of DNA with very short sequence motifs repeated in tandem; their often numerous alleles differ in the number of these repeat units. They are very common in eukaryotic DNA and can be amplified by the polymerase chain reaction, which allows the use of minute or degraded DNA samples. The alleles can be scored consistently and compared unambiguously, even across different gels.     

A Study of Polymorphisms in Three Loci Known to Influence Defensive Behavior Using PCR-SSCP and Direct Sequencing in the Iranian Honey Bee Population

Polymerase Chain Reaction (PCR) and subsequent separation of PCR products on polyacrylamide gels to find Single Stranded Conformation Polymorphisms (SSCP) was used for three loci, known from studies in North America to affect defensive behavior (Quantitative Trait Loci (QTL) sting1–3), on samples of Iranian honey bee (Apis mellifera L.). In the present study these loci were amplified with specific primers for sting1–3. The analysis of these loci using SSCP, created different patterns among samples. The polymorphisms observed in this study of the Iranian population of honey bees are a first step toward the eventual implementation of a genetic marker-based selection program to reduce defensive behavior. Now, because of this study, the markers are available to conduct more research so that the association of these polymorphic loci and aggressive traits in the Iranian honey bee population can be determined.     

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.     

Variability of six STR loci in populations from Central Spain.

Allele frequency distributions of six short tandem repeat (STR) loci, HUMTH01, HUMFES/FPS, HUMTPOX, HUMVWF/A31, HUMF13B, and HUMLPL, were determined in a population (101 individuals) of the Vera-Jerte region in West Central Spain. Amplified products were electrophoresed on denaturing polyacrylamide gels and silver-stained. The exact test demonstrated that none of the six loci deviated from Hardy-Weinberg equilibrium. R-matrix analysis in relation to other European samples agrees well with genetic distance results of Reynolds et al. (1983). Comparisons show that our population comes reasonably well within the range of variation of other European samples, and that the representation of these samples on the genetic map indicates a close relation between geographical location and distribution in the diagram.     

中华水韭(Isoetes sinensis)等位酶分析的初步研究

采用超薄平板聚丙烯酰胺等电聚焦技术对珍稀涉危植物中华水韭（Isoetes sinensis Palmer）72个样品的等位酶遗传变异进行了初步研究。从24种酶筛选,得到了13种酶系统可用于该植物的遗传变异分析,其中7种酶系统在中华水韭的迁地保护区群中检测出16个位点,共27个等位基因,12个位点表现出多态性。遗传多样性初步分析表明：中华水韭具较低的等位酶遗传多样性。研究结果为中华水韭的综合保育及居群恢复、重建提供了初步的依据。中华水韭遗传变异的全面研究将在进一步改善等位酶方法的基础上,采用其它更为灵敏的分子标记方法做深入检测。     

Rapid screening of a YAC library by pulsed-field gel Southern blot analysis of pooled YAC clones

A new method for screening of YAC libraries is described. Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis. A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each. Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs. Further purification was performed using standard colony hybridization procedures. Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these. Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters. The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments.     

药用植物天麻（Gastrodia elata Bl.）的等位酶遗传变异及其变型间的遗传关系

采用超薄平板聚丙烯酰胺等电聚焦电泳,对湖北西部药用植物天麻（Gastrodia elata Bl.）的4种变型进行了等位酶遗传变异的初步分析。在6个酶系统共检测到17个清晰位点和37个等位基因,其中多态位点11个,位点最大等位基因数为4,位点Acp-4和Prx-2的等位基因表现出变型特异性。遗传多样性分析结果表明：天麻在物种水平维持着较高遗传多样性,平均多态位点比率P=56.3%,每位点平均等位基因数A=2.1,平均预期杂合度He=0.222;但其变型水平遗传多样性较低（P=42.2%,A=1.7,He=0.146）,其中乌天麻遗传多样性最高,药天麻最低。天麻变型间遗传分化系数（GST）达0.3595,平均基因流（Nm）仅为0.4450,说明天麻变型间遗传分化较大,缺乏基因交流。聚类分析结果表明,天麻居群以变型为单元优先聚类而非地域优先聚类,揭示各天麻变型在遗传上是相对独立的进化显著单元,支持天麻种内变型的划分。红天麻与乌天麻两变型最为近缘,火天麻与药天麻两类群有着较近的遗传关系。     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.     

Linkage relationships and haplotype polymorphism among cichlid Mhc class II B loci.

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation.     

Search for genetic variation in the blood of Norwegian dairy goats reveals a new polymorphism at the HbβA locus

A total of 150 blood samples tested for serum albumin and transferrin and for red cell carbonic anhydrase, phosphoglucomutase, phosphogluconate dehydrogenase, phosphohexose isomerase, nucleoside phosphorylase, acid phosphatase, 'X'-protein and potassium concentration only showed variation at the 'X' protein and nucleoside phosphorylase loci. Isoelectric focusing over pH range 6-8 showed 145 samples to be of haemoglobin type A and 5 type AD. The haemoglobin A type was resolved into further types by separation over pH 6.9-7.5 in Immobiline polyacrylamide gels. A 2- or 4-band pattern was present in 136 of the samples; a genetic hypothesis based on four or more different haemoglobin A variants is proposed. 14 samples had a 3-, 5- or 6-band pattern. It is assumed that these are heterozygous for a variant of the II alpha gene.     

Detection, sequence patterns and function of unusual DNA structures.

Unusual DNA structures were detected by an electrophoretic procedure in which DNA fragments were separated according to size on agarose gels and then by shape on polyacrylamide gels. Fragments from yeast centromeres migrated faster in polyacrylamide than predicted from their base composition and size and this property was attributed to a nonrandom distribution of oligomeric A tracts that exhibited minima at 10-11 base intervals. Fragments from seven loci in 107 kb of DNA migrated anomalously slow and these fragments contained blocks of A2-6 in a 10-11 base periodicity which is indicative of bent DNA. The most pronounced bent sequences were found within yeast ARS1 and centered at 245 and 240 bp from the left and right ends of the adenovirus genome. Each sequence is approximately 150 bp away from a replication origin and the adenovirus sequences are within 50 bp of enhancers. Nuclear matrix attachment sites, which are also adjacent to enhancers, contain sequences characteristic of bent DNA. These results suggest that bent structures reside at the base of DNA loops in chromosomes.     

A comparison of two existing catalogs of the alleles of gliadin-coding loci in winter common wheat

Two catalogs of alleles of gliadin-coding loci, controlling synthesis of a storage protein of wheat caryopsis, gliadin, were compared. One catalog comprises the alleles detected according to the electrophoretic patterns in starch gels; the other, in polyacrylamide gels. Determination of the allelic state of gliadin-coding loci in 31 previously not studied cultivars of winter common wheat allowed us to construct a matching system for the alleles compiled in the two catalogs, which gives the possibility to compare the results of wheat cultivar analyses performed at different scientific institutions.     

A SINE-based dichotomous key for primate identification

For DNA samples or ‘divorced’ tissues, identifying the organism from which they were taken generally requires some type of analytical method. The ideal approach would be robust even in the hands of a novice, requiring minimal equipment, time, and effort. Genotyping SINEs (Short INterspersed Elements) is such an approach as it requires only PCR-related equipment, and the analysis consists solely of interpreting fragment sizes in agarose gels. Modern primate genomes are known to contain lineage-specific insertions of Alu elements (a primate-specific SINE); thus, to demonstrate the utility of this approach, we used members of the Alu family to identify DNA samples from evolutionarily divergent primate species. For each node of a combined phylogenetic tree (56 species; n = 8 [Hominids]; 11 [New World monkeys]; 21 [Old World monkeys]; 2 [Tarsiformes]; and, 14 [Strepsirrhines]), we tested loci (> 400 in total) from prior phylogenetic studies as well as newly identified elements for their ability to amplify in all 56 species. Ultimately, 195 loci were selected for inclusion in this Alu-based key for primate identification. This dichotomous SINE-based key is best used through hierarchical amplification, with the starting point determined by the level of initial uncertainty regarding sample origin. With newly emerging genome databases, finding informative retrotransposon insertions is becoming much more rapid; thus, the general principle of using SINEs to identify organisms is broadly applicable.     

A comparison of two existing catalogues of the alleles of gliadin-coding loci in winter common wheat

Two catalogs of alleles of gliadin-coding loci, controlling synthesis of a storage protein of wheat caryopsis, gliadin, were compared. One catalogue comprises the alleles detected according to the electrophoretic patterns in starch gels; the other, in polyacrylamide gels. Determination of the allelic state of gliadin-coding loci in 31 previously not studied cultivars of winter common wheat allowed us to construct a matching system for the alleles compiled in the two catalogs, which gives the possibility to compare the results of wheat cultivar analyses performed at different scientific institutions.     

Development and characterization of chloroplast microsatellite markers in Macaranga (Euphorbiaceae).

As part of our study on the phylogeography of the ant-plant genus Macaranga, we have screened for polymorphic regions in the chloroplast genome. Initially, ten universal PCR primer pairs targeted at chloroplast microsatellite loci were applied to a small set of specimens, covering various taxonomic levels from intrafamilial to intraspecific. Eight primer pairs produced PCR fragments that behaved as single and discrete bands on agarose gels. The five most promising candidate pairs were further analysed with an extended set of DNA templates, and PCR products were separated on sequencing gels. The number of size variants per locus varied from two to eight, combining into 17 haplotypes among 29 Macaranga accessions from 10 species. Comparative sequencing demonstrated that microsatellites were responsible for the observed size variation at three of five loci, whereas variation at the other loci was caused by larger insertions and (or) deletions (indels). In addition to poly(A) and poly(T) repeats, which are typically found in chloroplast DNA, we also identified a variable (CT)n repeat, with n = 4 to n = 8. Sequencing revealed three examples of size homoplasy, one of which was caused by a single base substitution that raised the actual number of haplotypes to 18. Relationships between haplotypes were assessed by phenetic analyses of size variants and by constructing a parsimony network based on sequence variation. For both types of analysis, the distribution of haplotypes correlated with geographically circumscribed regions rather than with taxonomic boundaries.     

System for DNA sequencing with resolution of up to 600 base pairs

A system capable of resolving about 500 bases is of interest for sequencing of longer DNA molecules. Studies on further optimization of resolution on DNA sequencing gels were carried out. The effect of physico-chemical properties of gels and buffers on resolution were tested, e.g. ionic strength and pH of buffers, different buffer systems, acrylamide concentration, crosslinker concentration, type of crosslinker, temperature of polymerization, denaturing conditions, gel length and thickness. Tested were as well different running conditions like electric field, gel temperature, dimension of sample slots. Gels 0.1-0.2 mm thick and up to 1.2 m long were cast and tested routinely. Gel lengths of 60-70 cm (for sequencing up to 350-400 bases) to about 100 cm (above 400 bases) are practicable. Little is gained in resolution by increasing the gel length from 1 to 1.2 m. Resolution was improved using 0.1 mm thick gels, at a higher pH value of 8.6-8.8, and molarity increased to 0.2 M. The sequencing pattern in the region of higher bases could be better resolved on a twice-magnified picture of that region on the autoradiogram. With the long gels (70-120 cm), it is advantageous to obtain the sequence overlap by running in parallel gels of different concentrations, without re-application of samples, all loaded at the same time. Buffer chamber for running of two of three gels and thermostating plates up to 1.2 m long were designed. In this way four to six thermostated gels can be run from a power supply with two inputs. Three 1 m long gels (concentrations: 4%, 6%, 12-16%) are loaded with several samples of DNA to be sequenced and run in parallel without re-application of the samples. With good samples, the sequence overlap from the gels could be counted up to 500 base pairs, with exceptionally good samples closer to 600 bases. At present this number seems to be near the limit of the resolving power of the polyacrylamide gels.     

Two-dimensional gel electrophoresis of proteins for genetic studies in douglas fir (Pseudotsuga menziesii)

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100°C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.This work was supported in part by Grant ATP PIRDES 508 444 from the INRA-CNRS.     

Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis-nitrocellulose blotting

An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria.     

Genetics of proteome variation for QTL characterization: application to drought-stress responses in maize

The proteome is emerging as an important concept of the post-genome era. Powerful nucleic acid approaches (EST, DNA chips, etc.) are still limited because DNA sequences and mRNA levels are not sufficient to predict the structure, function, amount, and activity of the proteins in the cell. The proteome can now be subjected to large-scale analysis, owing to spectacular progress in the techniques of identification of proteins excised from two-dimensional (2-D) gels. In addition, computer-based analysis of 2-D gels makes it possible to quantify the protein spot intensities, which are commonly genetically variable. The loci controlling these variations may be mapped on the genome (PQLs, Protein Quantity Loci). Beyond the interest for regulatory genetics and molecular biology, the PQL methodology can provide an additional tool for the difficult task of identifying QTLs (Quantitative Trait Loci), in the context of the candidate gene approach. The PQL methodology is presented with the example of the phosphoglycerate mutase variations in maize, and the candidate gene/protein approach is illustrated for traits responsive to drought stress.     

Analysis of polymorphism of three human Y-chromosome STR loci: DYS390, DYS392 and DYS393 in the population of northern Poland

The study aimed at development of a multiplex PCR system for amplification of three Y-chromosome STR loci: DYS390, DYS392 and DYS393, and its application in haplotype polymorphism analysis in the population of northern Poland. Due to interactions between originally published primers, a new DYS392 primer pair was proposed. In a population of 158 unrelated males, 28 different haplotypes could be observed, 12 of which were seen only once. The haplotype diversity is 0.805. Distribution of haplotypes of the studied loci is specific to the population of northern Poland and distinguishes it from compared West-European populations. To our knowledge, this is the first report on a Y-STR multiplex system that can be analysed on native polyacrylamide gels.