Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.     

Inheritance of microsatellite loci in the polyploid lake sturgeon (Acipenser fulvescens).

Inheritance in the expression of amplicons for four microsatellite primer pairs was determined using 10 families created from gametes of wild lake sturgeon (Acipenser fulvescens). Loci Afu34 and Afu68 expressed a maximum of two even-intensity bands per individual and had progeny genotype ratios that fit disomic inheritance (P > 0.05). Some variation exhibited at Afu34 and Afu68 was attributable to a null allele. Genotype expression at both loci also indicated that one female parent had transmitted unreduced gametes. Primer Afu39 amplified products that exhibited four gene doses, where genotype counts fit expected ratios for disomic inheritance (P > 0.05) indicating amplification of products from two disomic loci that share alleles. Meiotic drive was evident at the Afu39 loci based on a test for random segregation (P < 0.05). Only the expression of Afu19 gave evidence of tetrasomic inheritance based on a single progeny potentially produced by a double reduction gamete. No evidence for proposed octoploid inheritance was observed.     

Isolation and characterization of microsatellites in yellow perch (Perca flavescens)

A total of 45 microsatellite loci from yellow perch, Perca flavescens, were isolated and characterized. Among the 45 microsatellite loci, 32 had more than two alleles. A wild population of P. flavescens (n = 48) was used to examine the allele range of the microsatellite loci. Mendelian inheritance of alleles was confirmed by examining the amplified products in pair‐mated families. The number of alleles for the 32 polymorphic loci varied from two to 16, and observed heterozygosity ranged between 0.024 (YP79) and 0.979 (YP60). Cross‐species polymorphic amplification in four other Percidae species was successful for 22 loci.     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.     

A single base transversion in the flanking region of an equine microsatellite locus affects amplification of one allele

The equine dinucleotide microsatellite HMS7 is part of a microsatellite panel utilized in a parentage verification programme at the Veterinary Genetics Laboratory (Davis, California, USA). Apparent non-Mendelian inheritance was noted when a Quarter Horse mare was excluded as the parent of two offspring based on analysis of the HMS7 locus. The mare's DNA type qualified her as a parent of the offspring at an additional 20 microsatellite loci. The three animals appeared homozygous for HMS7 with each possessing an allele different from that of the other two animals. Polymerase chain reaction primers designed to bind outside the published primer-binding sites amplified an additional shared allele in all three horses, which qualified the mare as the dam of the two offspring. Sequencing of this newly detected allele revealed a C to A transversion in one of the published primer-binding regions. Apparent non-Mendelian inheritance at the HMS7 locus has been encountered in an additional 26 Quarter Horse parentage cases. In all instances, the lack of amplification and resultant 'null' allele was shown to be caused by the same transversion.     

The genetics of the sixth and seventh components of complement in the dog: Polymorphism,linkage, locus duplication,and silent alleles

The complement components C6 and C7 exhibit genetic polymorphism in the domestic dog. In the case of C6, there is a single locus with a null allele and two structural alleles; in the case of C7, there are two linked loci, each with three structural alleles. There is a null allele or locus deletion at one of these loci. In all cases, inheritance is autosomal and codominant. The C7 loci are closely linked to each other and to C6. This complex is not close to the dog major histocompatibility complex (MHC) locus.     

Genetic polymorphism in the bladder campion,Silene maritima

A survey of protein variability has been made in wild populations of Silene maritima, a perennial outcrossing coastal plant. In the most extensively studied populations, at Beesands, six out of 21 loci were polymorphic, and the average heterozygosity was 0.153. Four polymorphic loci were studied on an extensive shingle beach at Dungeness. Three loci showed no frequency cline with distance from the sea; a fourth locus, that for adenylate kinase, showed a barely significant cline, which was not confirmed by a repeat study in the same place a year later. Five polymorphic loci were studied at Beesands along a strip of shingle beach and up a contiguous cliff. No frequency difference between the two habitats was observed. There are significant differences in allelic frequencies between localities. It is concluded that the data fail to show an association between habitat and gene frequency, although they were collected in such a way as to be capable of demonstrating such an association if it existed.     

Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes, which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsp5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These classes of models are distinguished genetically by the fact that the first predicts that the output frequency of a given allele among the progeny of a large number of zygotes will approximately equal the average input frequency of that allele, while the second class predicts that any input bias will be amplified in the output. The data suggest that bias amplification does occur. We hypothesize that maternal inheritance of mitochondrial or chloroplast genes in many organisms may depend upon a biased input of organelle DNA molecules, which usually favors the maternal parent, followed by failure of the minority (paternal) molecules to replicate in many or all zygotes.     

Cross-species amplification, non-invasive genotyping, and non-Mendelian inheritance of human STRPs in Savannah baboons

Twenty-nine human microsatellite primer pairs were screened for their utility in the cross-species amplification of baboon DNA derived from both blood and feces as part of a larger study to identify paternal half sisters in a population of wild baboons (Papio cynocephalus). Forty-one percent (12/29) of the human primers successfully amplified baboon DNA. Of these 12 primers, six amplified fragments that were both polymorphic and heterozygous (mean number of alleles = 6, mean heterozygosity = 87%) and yielded repeatable results. However, only five of these six simple tandem repeat polymorphisms (STRPs) showed patterns of Mendelian inheritance (i.e., mothers and offspring shared at least one allele at each locus), and were therefore useful for determining relatedness between individuals. Analysis of the sixth primer revealed non-Mendelian inheritance, i.e., three of the six known mother-daughter pairs had no shared alleles. This failure was probably due to non-specific fragment amplification, and may have resulted from a different STRP locus being amplified in mother and daughter. This finding highlights the importance of sampling DNA from known parent-offspring pairs when screening microsatellite primers for genetic studies. Multiple, independent replications of genotypes and Mendelian checks are both particularly important when using cross-species amplification or when using a low-quality source of DNA.     

Chicken microsatellite primers are not efficient markers for Japanese quail

Domestic fowl or chicken (Gallus gallus) and Japanese quail (Coturnix japonica) belong to the family Phasianidae. The exchange of marker information between chicken and quail is an important step towards the construction of a high-resolution comparative genetic map in Phasianidae, which includes several poultry species of agricultural importance. We tested chicken microsatellite markers to see if they would be suitable as genetic linkage markers in Japanese quail. Twenty-six per cent (31/120) of chicken primers amplified individual loci in Japanese quail and 65% (20/31) of the amplified loci were found to be polymorphic. Eleven of the polymorphic loci were excluded as uninformative because of the lack of amplification in some individuals or high frequency of nonspecific amplification. The sequence information of the remaining nine loci revealed six of them to contain microsatellites that were nearly identical with those of the orthologous regions in chicken. For these six loci, allele frequencies were estimated in 50 unrelated quails. Although the very few chicken markers that do work well in quail could be used as anchor points for a comparative mapping, most chicken markers are not useful for studies in quail. Therefore, more effort should be committed to developing quail-specific markers rather than attempting to adapt chicken markers for work in quail.     

Isolation and characterization of polymorphic microsatellite loci from the Dungeness crab Cancer magister

The Dungeness crab, Cancer magister, is the focus of one of the most intensely harvested fisheries in North America. Given its economic importance, there is considerable interest in assessing the degree and spatial pattern of genetic structure in C. magister. To that end, we developed a series of 17 hypervariable microsatellite loci. Six of these 17 loci could be amplified in a single multiplex PCR reaction. Using dye‐labelled primers all six loci can be coamplified and scored simultaneously on an automated sequencer. The ability to multiplex multiple loci greatly increases the ease and speed of genotyping for this species.     

Microsatellite Variation in Two Populations of Free-Ranging Yellow Baboons (Papio hamadryas cynocephalus)

We investigated genetic variation at six microsatellite (simple sequence repeat) loci in yellow baboons (Papio hamadryas cynocephalus) at two localities: the Tana River Primate Reserve in eastern Kenya and Mikumi National Park, central Tanzania. The six loci (D1S158, D2S144, D4S243, D5S1466, D16S508, and D17S804) were all originally cloned from and characterized in the human genome. These microsatellites are polymorphic in both baboon populations, with the average heterozygosity across loci equal to 0.731 in the Tana River sample and 0.787 in the Mikumi sample. The genetic differentiation between the two populations is substantial. Kolmogornov–Smirnov tests indicate that five of the six loci are significantly different in allele frequencies in the two populations. The mean F _ST across loci is 0.069, and Shriver's measure of genetic distance, which was developed for microsatellite loci (Shriver et al., 1995), is 0.255. This genetic distance is larger than corresponding distances among human populations residing in different continents. We conclude that (a) the arrays of alleles present at these six microsatellite loci in two geographically separated populations of yellow baboons are quite similar, but (b) the two populations exhibit significant differences in allele frequencies. This study illustrates the potential value of human microsatellite loci for analyses of population genetic structure in baboons and suggests that this approach will be useful in studies of other Old World monkeys.     

STORM: software for testing hypotheses of relatedness and mating patterns

Storm is a software package that allows users to test a variety of hypotheses regarding patterns of relatedness and patterns of mate choice and/or mate compatibility within a population. These functions are based on four main calculations that can be conducted either independently or in the hypothesis-testing framework: internal relatedness; homozygosity by loci; pairwise relatedness; and a new metric called allele inheritance, which calculates the proportion of loci at which an offspring inherits a paternal allele different from that inherited from its mother. STORM allows users to test four hypotheses based on these calculations and Monte Carlo simulations: (i) are individuals within observed associations or groupings more/less related than expected; (ii) do observed offspring have more/less genetic variability (based on internal relatedness or homozygosity by loci) than expected from the gene pool; (iii) are observed mating pairs more/less related than expected if mating is random with respect to relatedness; and (iv) do observed offspring inherit paternal alleles different from those inherited from the mother more/less often than expected based on Mendelian inheritance.     

Evaluation of DNA pooling for the estimation of microsatellite allele frequencies: a case study using striped bass (Morone saxatilis)

Using striped bass (Morone saxatilis) and six multiplexed microsatellite markers, we evaluated procedures for estimating allele frequencies by pooling DNA from multiple individuals, a method suggested as cost-effective relative to individual genotyping. Using moment-based estimators, we estimated allele frequencies in experimental DNA pools and found that the three primary laboratory steps, DNA quantitation and pooling, PCR amplification, and electrophoresis, accounted for 23, 48, and 29%, respectively, of the technical variance of estimates in pools containing DNA from 2-24 individuals. Exact allele-frequency estimates could be made for pools of sizes 2-8, depending on the locus, by using an integer-valued estimator. Larger pools of size 12 and 24 tended to yield biased estimates; however, replicates of these estimates detected allele frequency differences among pools with different allelic compositions. We also derive an unbiased estimator of Hardy-Weinberg disequilibrium coefficients that uses multiple DNA pools and analyze the cost-efficiency of DNA pooling. DNA pooling yields the most potential cost savings when a large number of loci are employed using a large number of individuals, a situation becoming increasingly common as microsatellite loci are developed in increasing numbers of taxa.     

SSR cross-amplification and variation within coffee trees (Coffea spp.).

Primer sets were developed from 85 Coffea arabica sequences in addition to 25 already published primer sets. They were subsequently used for amplification in six African Coffea species: Coffea canephora (CAN), Coffea eugenioides (EUG), Coffea heterocalyx (HET), Coffea liberica (LIB), Coffea sp. Moloundou (MOL) and Coffea pseudozanguebariae (PSE). The amplification percentages for these 110 primer pairs ranged from 72.7% for LIB to 86.4% for PSE. Good transferability was thus obtained within the Coffea genus. When focusing on the two species CAN and PSE, high genetic diversity, high polymorphic locus rates (above 80%) and a mean allele number per polymorphic locus of more than 3 were noted. The estimated null allele percentage was -11% for PSE and -9% for CAN. Sixty three percent (CAN) and 79.5% (PSE) of the fixation index (Fis) values were positive. The within-species polymorphism information content (PIC) distribution showed two modes for both species. Although the two species shared 30 polymorphic loci, no correlation between CAN and PSE PIC values was obtained. All of these data are discussed in relation to the polymorphism level and the potential use of these SSRs for subsequent analysis of genetic diversity or genetic mapping.     

Polymorphic microsatellite DNA markers from the Oriental Fruit Fly Bactrocera dorsalis (Hendel)

Six polymorphic microsatellite loci are isolated from the Oriental fruit fly Bactrocera dorsalis (Hendel), an agricultural pest in Asia, including Taiwan. To assess their potential utility as high‐resolution genetic markers, polymerase chain reaction (PCR) primers, amplification conditions, and an automated fluorescence detection protocol were developed. In analyses of 71 individual flies from six different areas of Taiwan, allele numbers ranged from five to 25 were detected for each locus. The observed heterozygosity ranged between 0.268 and 0.737 among these loci. No linkage disequilibrium was found. These microsatellite markers have potential utility to population structure and gene flow studies of B. dorsalis (Hendel).     

The detection of nonhybrid, trisomic, and triploid offspring in sexual progeny of a mating of Phytophthora infestans.

Eighty single-oospore offspring of Phytophthora infestans from a mating of isolates, which had previously been analyzed for segregation of avirulence/virulence, were assessed for the inheritance of 20 RFLP markers. Three offspring were triploid; they inherited three alleles at all loci where this could be detected and when heterozygous, showed unequal intensities of hybridization with most probes. Twenty-four offspring were trisomic, as each had three doses of one or a few markers, evident from their inheritance of three alleles or from unequal hybridization to one probe. Coinheritance of the extra allele(s) and mitochondrial haplotype in the majority of trisomic offspring suggested that meiosis in oogonia was more aberrant than in antheridia. Linkage analysis was performed on 50 offspring, which were assumed to be euploid; six small linkage groups were detected and several avirulence loci were found to be linked. The origins of aberrant offspring are discussed.     

Isolation of twenty low stutter di- and tetranucleotide microsatellites for population analyses of walleye pollock and other gadoids

Fourteen tetra- and six dinucleotide microsatellites, which exhibit minimal stuttering following amplification via PCR were developed from walleye pollock Theragra chalcogramma . Most of these loci were isolated from a library enriched for tetranucleotide microsatellites by hybridization of genomic DNA to (gata)₇ oligonucleotides bound to streptavidin-coated paramagnetic beads. The average heterozygosity of these loci is ∼80%, and ranges from 53–95%. Mendelian inheritance was confirmed in five families, each consisting of a minimum of 10 or more offspring. Primer sets for all 20 loci were also evaluated in Arctic cod Boreogadus saida , Pacific cod Gadus macrocephalus , Pacific tomcod Microgadus proximus , sa.ron cod Eleginus gracilis , Pacific hake Merluccius productus , Atlantic cod Gadus morhua , haddock Melanogrammus aeglefinus , blue whiting Micromesistius poutassou , and European hake Merluccius merluccius . In each of these species, 3–19 primer sets amplified variable microsatellite loci. These loci, which exhibit little stutter and moderate to high variability, should be useful population markers in pollock and other gadoid fishes.     

Nuclear DNA restriction site polymorphisms and the phylogeny and population structure of an intertidal snail species complex (Littorina)

Primers for amplification of four novel, unlinked nuclear DNA loci, the first reported for the rough periwinkles of the genus Littorina, are described. Patterns of restriction site polymorphism for these loci are detailed within the rough periwinkles. RFLPs are not found to be diagnostic for any of the currently accepted species within this group, nor for any of the contentious subspecies, or forms, whose taxonomic status is uncertain. However, there are important differences in allele frequencies between these taxa and certain of these mirror differences detected in a previous study of the mitochondrial DNA. These allele frequency data are used to construct a phylogeny in which groupings of the three recognised species are obvious when either Nei's genetic distances or Reynold's distances are clustered. Contentious forms (L. neglecta, L. saxatilis 'b' and L. tenebrosa) do not cluster as distinct taxa, although populations of L. neglecta have important allele frequency differences from L. saxatilis. These four loci have confirmed the consensus view of Littorina phylogeny and provided important information on population structure-however four loci is insufficient for reaching definitive conclusions. Since analysis of nuclear DNA polymorphisms such as these is invaluable for analysis of phylogeny, population structure and phylogeography, identification of additional loci is considered imperative.     

Human minisatellite alleles detectable only after PCR amplification.

We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.