Microsatellite polymorphism and its association with body weight and selected morphometrics of farm red fox (Vulpes vulpes L.)

Polymorphism of 30 canine-derived microsatellites was studied in a group of 200 red foxes kept on 2 Polish farms. 22 out of 30 microsatellites were selected to study association between marker genotypes and body weight (BW), body length (BL), body circumference (BC), tail length (TL), ear height (EH), length of the right front limb (FRLL), length of the right rear limb (RRLL), length of the right front foot (FRFL) and length of the right rear foot (RRFL). A total of 112 alleles and 243 genotypes were found at 22 autosomal microsatellite loci. Three monomorphic loci deemed as uninformative were excluded from the study. The association between marker genotypes and the studied traits was analysed using general linear model (GLM) procedure and least squares means (LSM). Linkage disequilibrium (LD) was estimated to assess non-random association between microsatellite loci. Out of 19 microsatellites studied four markers showed no association with the studied traits, three markers had a significant effect on one trait, and another three markers had significant effect on two traits. Among ten microsatellites with significant effect on four economically important traits (BW, BL, BC, TL) four were associated with two characters: marker FH2613 with BW and BC, marker FH2097withBL and BC, marker ZUBECA6 with BW and BC, whereas marker REN75M10 was associated with BL and TL. The strongest LD (r² ranged from 0.15 to 0.33) was estimated between nine loci with significant effect on economically important traits (BW, BL, BC, TL).     

Genetic variation of three autosomal STR Loci D21S1435, D21S1411, and D21S1412 in Korean population

The autosomal tetranucleotide short tandem repeat loci D21S1435, D21S1411 and D21S1412 were analyzed in samples of unrelated 200 Korean individuals. The loci showed no significant deviations from Hardy–Weinberg equilibrium. Alleles were assigned according to the International Society for Forensic Haemogenetics (ISFH) recommendations. The power of discrimination of the analyzed markers was found to be high for the populations, thereby facilitating the validation and efficiency of these STR markers in forensic human identification and paternity testing. To our knowledge, this is the first report of the nomenclature and the allele frequency data for these three STR loci in Korean population.     

A proposal for standardization in forensic bovine DNA typing: allele nomenclature of 16 cattle-specific short tandem repeat loci

In this study, a proposal is presented for the allele nomenclature of 16 polymorphic short tandem repeat (STR) loci ( BM1824 , BM2113 , ETH10 , ETH225 , INRA023 , SPS115 , TGLA122 , TGLA126 , TGLA227 , ETH3 , TGLA53 , BM1818 , CSRM60 , CSSM66 , HAUT27 and ILSTS006 ) for bovine genotyping ( Bos taurus ). The nomenclature is based on sequence data of the polymorphic region(s) of the STR loci as recommended by the DNA commission of the International Society of Forensic Genetics for human DNA typing. To cover commonly and rarely occurring alleles, a selection of animals homozygous for the alleles at these STR loci were analysed and subjected to sequence studies. The alleles of the STR loci consisted either of simple or compound dinucleotide repeat patterns. Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats including all variable regions within the amplified fragment. The set of 16 STR markers should be propagated for the use in all bovine applications including forensic analysis.     

An integrative AmpSeq platform for highly multiplexed marker-assisted pyramiding of grapevine powdery mildew resistance loci

Resistance breeding often requires the introgression and tracking of resistance loci from wild species into domesticated backgrounds, typically with the goal of pyramiding multiple resistance genes, to provide durable disease resistance to breeding selections and ultimately cultivars. While molecular markers are commonly used to facilitate these efforts, high genetic diversity and divergent marker technologies can complicate marker-assisted breeding strategies. Here, amplicon sequencing (AmpSeq) was used to integrate SNP markers with dominant presence/absence markers derived from genotyping-by-sequencing and other genotyping technologies, for the simultaneous tracking of five loci for resistance to grapevine powdery mildew. SNP haploblocks defined the loci for REN1, REN2 and REN3, which confer quantitative resistance phenotypes that are challenging to measure via field ratings of natural infections. Presence/absence markers for RUN1 and REN4 were validated to predict qualitative resistance phenotypes and corresponded with previous presence/absence fluorescent electrophoretic assays. Thus, 37 AmpSeq-derived markers were identified for the five loci, and markers for REN1, REN2, REN4 and RUN1 were used for multiplexed screening and selection within diverse breeding germplasm. Poor transferability of SNP markers indicated imperfect marker-trait association in some families. Together, AmpSeq SNP haploblocks and presence/absence markers provide a high-throughput, cost-effective tool to integrate divergent technologies for marker-assisted selection and genetic analysis of introgressed disease resistance loci in grapevine.     

Linkage of the locus for canine dewclaw to chromosome 16

The canine species, including wolf and jackal, have four digits on the hind limb. It was thought that an extra first digit on the hind limb, named dewclaw, was a hereditary defect. For genetically related canine pedigrees with 73 members with dewclaws, we carried out a genome-wide scan for linkage with microsatellites. With an assumption of autosomal dominant mode of inheritance, significant linkages were detected for the markers on canine chromosome 16. The maximum two-point lod score of 20.76 was obtained for the REN85M08/REN85N14 markers at a recombination fraction of 0.00. For efficient analysis of linkage, a revised order of the chromosomal markers was established by assigning all the existing markers from the previous linkage and radiation hybrid maps. A chromosome-wide haplotype analysis revealed the location of the dewclaw locus within a few centimorgan intervals delimited by the UCMCF12 and CXX876 markers. Canine chromosome 16 is known to have syntenic relationships with human chromosomes 4q, 7q, and 8p.     

Extending a RFLP-based genetic map of rye using random amplified polymorphic DNA (RAPD) and isozyme markers

RFLP-based genetic map of rye, developed previously using a cross of lines DS2×RXL10 (F₂ generation), was extended with 69 RAPD and 12 isozyme markers. The actual map contains 282 markers dispersed on all seven chromosomes and spans a distance of 1,140 cM. The efficiency of mapping RAPD markers was close to ten loci per 100-screened arbitrary primers. A strong selection of polymorphic, intensive and reproducible fragments was necessary to reveal individual marker loci that could be assigned to rye chromosomes. Newly mapped markers cover a substantial part of the rye genome and constitute a valuable tool suitable for map saturation, marker-aided selection and phenetic studies. A specific nomenclature for the RAPD loci mapped on individual rye chromosomes, which could be helpful in managing of accumulating data, is proposed. Received: 8 May 2000 / Accepted: 17 October 2000     

Instability of the minisatellite sequence in the first intron of the rat renin gene and localization of the gene to chromosome 13q13 between FH and PEPC loci.

A minisatellite sequence in the first intron of the rat renin gene showed five-allelic polymorphism in 11 inbred rat strains. A new allelic variant, which was thought to be generated in the germ line, was observed in 136 animals of two sets of backcross progenies originating from parental strains with different alleles. These facts suggested that the minisatellite is genetically unstable. A linkage analysis using the backcross progenies confirmed the assignment of renin locus (REN) on linkage group (LG) X at a site between FH (fumarate hydratase) and PEPC (peptidase) loci. Fluorescence in situ hybridization allowed mapping of the renin gene on rat chromosome 13q13.     

Microsatellite characterization of Cimarron Uruguayo dogs

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each microsatellite, the genetic variability and the consanguinity were calculated, as were the polymorphic information content (PIC) and the probability of exclusion (PE). All of the microsatellites studied were polymorphic. FH 2361, FH 2305 and PEZ 03 were the most informative, with PIC values > 0.7, in agreement with results for other canine breeds. The PE values for the markers were within the ranges previously described and were generally greater for microsatellites with higher PIC values. The heterozygosity value (0.649) was considered high since only nine microsatellites were analyzed. Compared with data for other breeds, the results obtained here indicate that Cimarron Uruguayo dogs have high genetic diversity.     

Comparison of microsatellites and amplified fragment length polymorphism markers for parentage analysis

This study compares the properties of dominant markers, such as amplified fragment length polymorphisms (AFLPs), with those of codominant multiallelic markers, such as microsatellites, in reconstructing parentage. These two types of markers were used to search for both parents of an individual without prior knowledge of their relationships, by calculating likelihood ratios based on genotypic data, including mistyping. Experimental data on 89 oak trees genotyped for six microsatellite markers and 159 polymorphic AFLP loci were used as a starting point for simulations and tests. Both sets of markers produced high exclusion probabilities, and among dominant markers those with dominant allele frequencies in the range 0.1-0.4 were more informative. Such codominant and dominant markers can be used to construct powerful statistical tests to decide whether a genotyped individual (or two individuals) can be considered as the true parent (or parent pair). Gene flow from outside the study stand (GFO), inferred from parentage analysis with microsatellites, overestimated the true GFO, whereas with AFLPs it was underestimated. As expected, dominant markers are less efficient than codominant markers for achieving this, but can still be used with good confidence, especially when loci are deliberately selected according to their allele frequencies.     

On the distribution of temporal variations in allele frequency: consequences for the estimation of effective population size and the detection of loci undergoing selection

The effective population size (Ne) is frequently estimated using temporal changes in allele frequencies at neutral markers. Such temporal changes in allele frequencies are usually estimated from the standardized variance in allele frequencies (Fc). We simulate Wright-Fisher populations to generate expected distributions of Fc and of Fc (Fc averaged over several loci). We explore the adjustment of these simulated Fc distributions to a chi-square distribution and evaluate the resulting precision on the estimation of Ne for various scenarios. Next, we outline a procedure to test for the homogeneity of the individual Fc across loci and identify markers exhibiting extreme Fc-values compared to the rest of the genome. Such loci are likely to be in genomic areas undergoing selection, driving Fc to values greater (or smaller) than expected under drift alone. Our procedure assigns a P-value to each locus under the null hypothesis (drift is homogeneous throughout the genome) and simultaneously controls the rate of false positive among loci declared as departing significantly from the null. The procedure is illustrated using two published data sets: (i) an experimental wheat population subject to natural selection and (ii) a maize population undergoing recurrent selection.     

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

 The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories. Received: 10 May 1997 / Revised: 1 August 1997     

A novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration.

Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.     

Variation of short tandem repeats within and between species belonging to the Canidae family

Frequency distribution and allele size in 20 canine microsatellite loci were analyzed in 33 flat-coated retrievers, 32 dachshunds, 10 red foxes, and 10 Arctic foxes. Overall, the major difference between the two dog breeds was the relative allele frequencies rather than the size ranges of alleles at the individual locus. The average heterozygosity within the two dog breeds was not significantly different. Since the average heterozygosity at several polymorphic loci is a relative measure of heterogeneity within the population, analysis of heterozygosity within microsatellite loci is suggested as a measure for the diversity of populations. Eighty percent (16 of 20) of the canine microsatellite primer pairs amplified corresponding loci in the two fox species. This reflects a very high sequence conservation within the Canidae family relative to findings in, for instance, the Muridae family. This indicates that it will be possible to utilize the well-characterized fox karyotype instead of the dog karyotype as a step towards physical mapping of the dog genome. Analysis of exclusion power and probabilities of genetic identity between unrelated animals by use of the seven most informative loci demonstrated that it will be possible to assemble a panel of microsatellite loci that is effective for parentage analysis in all breeds.     

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.     

A proposal for standardization in forensic equine DNA typing: allele nomenclature for 17 equine‐specific STR loci

In this study, a proposal is presented for the allele nomenclature of 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) for equine genotyping (Equus caballus). The nomenclature is based on sequence data of the polymorphic region of the STR loci as recommended by the DNA commission of the International Society for Forensic Genetics for human DNA typing. For each STR locus, several alleles were selected and animals homozygous for those alleles were subjected to sequence analysis. The alleles of the 17 STR loci consisted either of simple (10), compound (6) or complex repeat patterns (1). Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats, including all variable regions within the amplified fragment.     

Semiautomated and simultaneous analysis of the interleukin-10 gene microsatellites IL-10G and IL-10R by fluorescence-based polymerase chain reaction reveals significant differences in allele distributions between Caucasians (Germany) and Africans (Gabon)

Interleukin-10 (IL-10) is an important immunoregulatory cytokine influencing many aspects of the adaptive and inflammatory immune response. Two dinucleotide repeats have been identified in the 5'-UTR of IL-10 and shown to be useful genetic markers in several diseases. A simple, two-colour fluorescence assay was developed for determination of microsatellite fragment length by an automatic sequencer. Using this method polymorphisms at the IL-10G and IL-10R loci of the 5' flanking region of the IL-10 gene can be identified simultaneously. A unified standard nomenclature was applied to the known IL-10G and IL-10R microsatellites. The multiplex PCR approach was used to compare the allele frequencies in two independent donor groups from Germany (Caucasian), comprising 112 and 106 cases, respectively, and one group from Gabon (African) including 91 donors. Significant differences in the allele distribution were found. Both Caucasian populations tested showed no significant differences in their allele and genotype distribution. Whereas in Africans, allele IL-10G25 is rare at 3% compared to 21% in Caucasian, alleles IL-10G22 and G23 are more prevalent in Africans than in Caucasians (22% versus 10% and 26% versus 7%, respectively). Within the IL-10R locus, the allele R13 was observed at 88% in the African group compared to 69% in Caucasians. These data may help immunogenetic studies in diseases, where IL-10 is thought to be deregulated.     

Detecting population growth, selection and inherited fertility from haplotypic data in humans

The frequency of a rare mutant allele and the level of allelic association between this allele and one or several closely linked markers are frequently measured in genetic epidemiology. Both quantities are related to the time elapsed since the appearance of the mutation in the population and the intrinsic growth rate of the mutation (which may be different from the average population growth rate). Here, we develop a method that uses these two kinds of genetic data to perform a joint estimation of the age of the mutation and the minimum growth rate that is compatible with its present frequency. In absence of demographic data, it provides a useful estimate of population growth rate. When such data are available, contrasts among estimates from several loci allow demographic processes, affecting all loci similarly, to be distinguished from selection, affecting loci differently. Testing these estimates on populations for which data are available for several disorders shows good congruence with demographic data in some cases whereas in others higher growth rates are obtained, which may be the result of selection or hidden demographic processes.     

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.     

Linkage of Van der Woude syndrome (VWS) to REN and exclusion of the candidate gene TGFB2 from the disease locus in a large pedigree

Van der Woude syndrome (VWS) is an autosomal dominant disorder associated with one or more of the following features: clefting of the primary or secondary palate, hypodontia or lower lip pits. It has been estimated to account for 2% of all cases of cleft lip and palate. VWS is one of the rare disorders in which clefting of the primary and secondary palate may be seen to segregate as components associated with the same gene. Because of its autosomal dominant inheritance, VWS is readily accessable to linkage analysis as a preliminary step in the identification of the molecular abnormality underlying the clefting effect in the primary and secondary palate. A reported linkage between REN and VWS has promoted us to use pHRnX3.6 (REN) and several markers surrounding REN for a linkage analysis in a large Swiss family. In a second step, linkage analysis was performed to study restriction fragment length polymorphisms for the candidate gene TGFB2 and other loci recently mapped to the candidate region 1q32–1q41. Evidence for linkage ( = 0.00, lod score = 3.01) between REN and VWS could be confirmed in this pedigree. TGFB2 demonstrated recombination with the disease locus and is unlikely to be causative in VWS. The results of a multipoint linkage analysis showed that VWS was flanked by D1S65 and TGFB2 at a maximum location score of 20.3.     

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain

Abstract Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.