Novel algorithm for automated genotyping of microsatellites

Microsatellites or short tandem repeats (STRs) are abundant in the human genome with easily assayed polymorphisms, providing powerful genetic tools for mapping both Mendelian and complex traits. Microsatellite genotyping requires detection of the products of polymerase chain reaction (PCR) amplification by electrophoresis, and analysis of the peak data for discrimination of the true allele. A high-throughput genotyping approach requires computer-based automation at both the detection and analysis phases. In order to achieve this, complicated peak patterns from individual alleles must be interpreted in order to assign alleles. Previous methods consider limited types of noise peaks and cannot provide enough accuracy. By pattern recognition of various types of noise peaks, such as stutter peaks and additional peaks, we have achieved an overall average accuracy of 94% for allele calling in our actual data. Our algorithm is crucial for a high-throughput genotyping system for microsatellite markers by reducing manual editing and human errors.     

A novel method for automatic genotyping of microsatellite markers based on parametric pattern recognition

Genetic mapping of loci affecting complex phenotypes in human and other organisms is presently being conducted on a very large scale, using either microsatellite or single nucleotide polymorphism (SNP) markers and by partly automated methods. A critical step in this process is the conversion of the instrument output into genotypes, both a time-consuming and error prone procedure. Errors made during this calling of genotypes will dramatically reduce the ability to map the location of loci underlying a phenotype. Accurate methods for automatic genotype calling are therefore important. Here, we describe novel algorithms for automatic calling of microsatellite genotypes using parametric pattern recognition. The analysis of microsatellite data is complicated both by the occurrence of stutter bands, which arise from Taq polymerase misreading the number of repeats, and additional bands derived form the non-template dependent addition of a nucleotide to the 3 end of the PCR products. These problems, together with the fact that the lengths of two alleles in a heterozygous individual may differ by only two nucleotides, complicate the development of an automated process. The novel algorithms markedly reduce the need for manual editing and the frequency of miscalls, and compares very favourably with commercially available software for automatic microsatellite genotyping.     

Guidelines for genotyping in genomewide linkage studies: single-nucleotide-polymorphism maps versus microsatellite maps

Genomewide linkage scans have traditionally employed panels of microsatellite markers spaced at intervals of approximately 10 cM across the genome. However, there is a growing realization that a map of closely spaced single-nucleotide polymorphisms (SNPs) may offer equal or superior power to detect linkage, compared with low-density microsatellite maps. We performed a series of simulations to calculate the information content associated with microsatellite and SNP maps across a range of different marker densities and heterozygosities for sib pairs (with and without parental genotypes), sib trios, and sib quads. In the case of microsatellite markers, we varied density across 11 levels (1 marker every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cM) and marker heterozygosity across 6 levels (2, 3, 4, 5, 10, or 20 equally frequent alleles), whereas, in the case of SNPs, we varied marker density across 4 levels (1 marker every 0.1, 0.2, 0.5, or 1 cM) and minor-allele frequency across 7 levels (0.5, 0.4, 0.3, 0.2, 0.1, 0.05, and 0.01). When parental genotypes were available, a map consisting of microsatellites spaced every 2 cM or a relatively sparse map of SNPs (i.e., at least 1 SNP/cM) was sufficient to extract most of the inheritance information from the map (>95% in most cases). However, when parental genotypes were unavailable, it was important to use as dense a map of markers as possible to extract the greatest amount of inheritance information. It is important to note that the information content associated with a traditional map of microsatellite markers (i.e., 1 marker every ~10 cM) was significantly lower than the information content associated with a dense map of SNPs or microsatellites. These results strongly suggest that previous linkage studies that employed sparse microsatellite maps could benefit substantially from reanalysis by use of a denser map of markers.     

Index, Comprehensive Microsatellite, and Unified Linkage Maps of Human Chromosome 14 with Cytogenetic Tie Points and a Telomere Microsatellite Marker

Three sets of linkage maps (index, comprehensive microsatellite, and unified) have been constructed for human chromosome 14 based on genotypes from the CEPH reference pedigrees. The index maps consist of 18 microsatellite markers, with heterozygosities of at least 68% and intermarker spacing no greater than 11 cM. The sex-average comprehensive microsatellite map is 125 cM in length and includes 115 markers with 54 loci uniquely placed with odds for marker order of at least 1000:1. The sex-average index map length is 121 cM, and the female- and male-specific maps are 143 and 101 cM, respectively. A unified map was also constructed from 147 loci (162 marker systems), which includes 32 RFLP markers in addition to the 115 microsatellites. The sex-average length of the unified map is 128 cM with 69 loci uniquely placed. Our maps are anchored by a microsatellite telomere marker sCAW1 (D14S826), developed from a telomere YAC clone TYAC196, which extends the linkage map to the physical terminus of the long arm of chromosome 14. Furthermore, we have also physically mapped seven of the loci by fluorescencein situhybridization of cosmid clones orAlu-PCR products amplified from YACs containing the marker sequences. Together with previously established cytogenetic map designations for other loci, our maps display links between genetic markers for 10 of 13 cytogenetic bands of chromosome 14 at the 550 genome band resolution.     

Isolation and characterization of seven tetranucleotide microsatellite loci in mungbean,Vigna radiata

A simple and rapid method for isolating tetranucleotide microsatellites in mungbean, Vigna radiata, based on the 5′‐anchored polymerase chain reaction technique, revealed 15 microsatellite sequences. We report on the characterization of seven polymorphic microsatellite loci in V. radiata. The number of alleles per locus ranged from 2 to 6, whereas the observed heterozygosity ranged from 0 to 0.561. Tetranucleotide markers are useful because they amplify fewer stutter bands thus making scoring easier. These markers will be useful for detecting genetic variation in mungbean varieties for germplasm management and development of the crop.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

A PCR-Based Genetic Map for Human Chromosome 3

Oligonucleotide primers for 125 simple sequence repeat microsatellite-based genetic markers have been assayed by polymerase chain reaction (PCR) in the CEPH reference family panel. These microsatellites include 101 dinucleotide repeats as well as 24 new tetranucleotide repeats. The average heterozygosity of this marker set was 72.4%. Genetic data were analyzed with the genetic mapping package LINKAGE. A subset of these microsatellite markers define a set of 56 uniquely ordered loci (>1000:1 against local inversion) that span 271 cM. Sixty-seven additional loci were tightly linked to markers on the uniquely ordered map, but could not be ordered with such high precision. These markers were positioned by CMAP into confidence intervals. One hundred thirteen of the microsatellite markers were also tested on a chromosome 3 framework somatic cell hybrid panel that divides this chromosome into 23 cytogenetically defined regions, integrating the genetic and physical maps of this chromosome. The high density, high heterozygosity, and PCR format of this genetically and physically mapped set of markers will accelerate the mapping and positional cloning of new chromosome 3 genes.     

A collection of ordered tetranucleotide-repeat markers from the human genome. The Utah Marker Development Group.

A collection of 1,069 human PCR-based genetic markers has been developed, and their distribution over the 22 autosomes and the X chromosome has been determined. Each marker was developed around a short-tandem-repeat DNA sequence. The majority (85%) of the markers described here were selected to contain tetranucleotide repeats, because these repeats show better stability during PCR than do dinucleotide repeats. Linkage maps constructed from genotypes collected with these markers in four CEPH pedigrees (1331, 1332, 1362, and 884) covered 3,417 cM of the human genome. More than 600 of the loci revealed heterozygosities > .70. Overall, 444 loci were ordered, with odds > 100:1 against inversion of adjacent loci. The average distance between markers was 7.4 cM on the autosomes and 24.8 cM on the X chromosome. Likely locations (100:1 odds intervals) were assigned for the remaining 621 short-tandem-repeat polymorphisms, as well as for 160 other markers that are present on the framework maps published by the Cooperative Human Linkage Center. Four markers specific to the Y chromosome are also reported here. From our maps, 347 markers were chosen to define "index" maps for each of the 22 autosomes. The index markers detect loci with an average heterozygosity of .85 and cover 3,169 cM of the autosomes, with an average distance between markers of 9.2 cM. These polymorphic short tandem repeats will be highly useful as reagents for the ongoing genetic and physical mapping of the human genome and for characterization of genetic changes in cancer.     

Toward fully automated genotyping: allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy.

Human genetic maps have made quantum leaps in the past few years, because of the characterization of > 2,000 CA dinucleotide repeat loci: these PCR-based markers offer extraordinarily high PIC, and within the next year their density is expected to reach intervals of a few centimorgans per marker. These new genetic maps open new avenues for disease gene research, including large-scale genotyping for both simple and complex disease loci. However, the allele patterns of many dinucleotide repeat loci can be complex and difficult to interpret, with genotyping errors a recognized problem. Furthermore, the possibility of genotyping individuals at hundreds or thousands of polymorphic loci requires improvements in data handling and analysis. The automation of genotyping and analysis of computer-derived haplotypes would remove many of the barriers preventing optimal use of dense and informative dinucleotide genetic maps. Toward this end, we have automated the allele identification, genotyping, phase determinations, and inheritance consistency checks generated by four CA repeats within the 2.5-Mbp, 10-cM X-linked dystrophin gene, using fluorescein-labeled multiplexed PCR products analyzed on automated sequencers. The described algorithms can deconvolute and resolve closely spaced alleles, despite interfering stutter noise; set phase in females; propagate the phase through the family; and identify recombination events. We show the implementation of these algorithms for the completely automated interpretation of allele data and risk assessment for five Duchenne/Becker muscular dystrophy families. The described approach can be scaled up to perform genome-based analyses with hundreds or thousands of CA-repeat loci, using multiple fluorophors on automated sequencers.     

A genetic linkage map of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>): sex-linked microsatellite markers and high recombination rates

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.     

Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.)

This paper aims at providing reliable and cost effective genotyping conditions, level of polymorphism in a range of genotypes and map position of newly developed microsatellite markers in order to promote broad application of these markers as a common set for genetic studies in pea. Optimal PCR conditions were determined for 340 microsatellite markers based on amplification in eight genotypes. Levels of polymorphism were determined for 309 of these markers. Compared to data obtained for other species, levels of polymorphism detected in a panel of eight genotypes were high with a mean number of 3.8 alleles per polymorphic locus and an average PIC value of 0.62, indicating that pea represents a rather polymorphic autogamous species. One of our main objectives was to locate a maximum number of microsatellite markers on the pea genetic map. Data obtained from three different crosses were used to build a composite genetic map of 1,430 cM (Haldane) comprising 239 microsatellite markers. These include 216 anonymous SSRs developed from enriched genomic libraries and 13 SSRs located in genes. The markers are quite evenly distributed throughout the seven linkage groups of the map, with 85% of intervals between the adjacent SSR markers being smaller than 10 cM. There was a good conservation of marker order and linkage group assignment across the three populations. In conclusion, we hope this report will promote wide application of these markers and will allow information obtained by different laboratories worldwide in diverse fields of pea genetics, such as QTL mapping studies and genetic resource surveys, to be easily aligned.Electronic Supplementary Material Supplementary material is available for this article at     

Dinucleotide Repeat Loci Contribute Highly Informative Genetic Markers to the Human Chromosome 2 Linkage Map

Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene (Hoth and Engel, 1991). These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers.     

Second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (Felis catus)

We report construction of second-generation integrated genetic linkage and radiation hybrid (RH) maps in the domestic cat (Felis catus) that exhibit a high level of marker concordance and provide near-full genome coverage. A total of 864 markers, including 585 coding loci (type I markers) and 279 polymorphic microsatellite loci (type II markers), are now mapped in the cat genome. We generated the genetic linkage map utilizing a multigeneration interspecies backcross pedigree between the domestic cat and the Asian leopard cat (Prionailurus bengalensis). Eighty-one type I markers were integrated with 247 type II markers from a first-generation map to generate a map of 328 loci (320 autosomal and 8 X-linked) distributed in 47 linkage groups, with an average intermarker spacing of 8 cM. Genome coverage spans approximately 2,650 cM, allowing an estimate for the genetic length of the sex-averaged map as 3,300 cM. The 834-locus second-generation domestic cat RH map was generated from the incorporation of 579 type I and 255 type II loci. Type I markers were added using targeted selection to cover either genomic regions underrepresented in the first-generation map or to refine breakpoints in human/feline synteny. The integrated linkage and RH maps reveal approximately 110 conserved segments ordered between the human and feline genomes, and provide extensive anchored reference marker homologues that connect to the more gene dense human and mouse sequence maps, suitable for positional cloning applications.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

Ten novel microsatellite markers for the freshwater sleeper Micropercops swinhonis (Günther, 1873), with testing of cross‐species amplification in two other fishes from suborder Gobioidei

Ten novel polymorphic microsatellite loci were developed for the freshwater sleeper Micropercops swinhonis, a relatively abundant and broadly distributed lake‐dwelling fish in Asia, using next generation sequencing. These markers were tested in a population of 48 individuals, and characteristics suggest that they may be useful for population genetic studies. The average number of alleles per marker was 6.6 (range: 2–26), the average expected heterozygosity was 0.55 (range: 0.081–0.955), and the average polymorphic information content value per marker was 0.507 (range: 0.077–0.942). No marker showed significant deviation from Hardy‐Weinberg Equilibrium following Bonferroni correction of significance values, and no markers showed evidence of null alleles, large allele dropout, or scoring errors from stutter bands. Two markers had successful amplification in Odontobutis potamophila, and one marker had successful amplification in Rhinogobius giurinus. These novel markers may be useful for further research in population genetic studies involving M. swinhonis.     

Genetic mapping of three alleles at the Pm3 locus conferring powdery mildew resistance in common wheat (Triticum aestivum L.).

A set of differential isolates of Blumeria graminis f.sp. tritici was used to identify 10 alleles at the Pm3 locus on the short arm of chromosome 1A. Three F3 populations were used to map Pm3h in Abessi, Pm3i in line N324, and Pm3j alleles in GUS 122 relative to microsatellite markers. In total, 13 marker loci were mapped on chromosome 1AS and 1 marker on 1AL. The order of marker loci in the 3 mapping populations is consistent with previously published maps. All 3 alleles were mapped in the distal region of chromosome 1AS. The present study indicated that microsatellite markers are an ideal marker system for comparative mapping of alleles at the same gene locus in different mapping populations. The linkage distances of the closest microsatellite marker, Xgwm905-1A, to Pm3h, Pm3i, and Pm3j were 3.7 cM, 7.2 cM, and 1.2 cM, respectively. The microsatellite marker Xgwm905-1A cannot be used to distinguish between Pm3 alleles. The development of specific markers for individual Pm3 alleles is discussed on the basis of the recently cloned Pm3b allele.     

Genetic Analysis of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Using Microsatellite Markers

Microsatellites are repetitive genomic elements that show high levels of variation and therefore are useful tools for studying genetic polymorphism and constructing genetic linkage maps of eukaryotic organisms. Porphyra yezoensis Ueda is an economically important seaweed that is being targeted for genetic improvement using marker-assisted breeding. Hence, in an attempt to develop microsatellite markers for P. yezoensis, a microsatellite (or simple sequence repeat)-enriched library was constructed to identify (GA)n and (CA)n motifs. A total of 71 perfect microsatellite clones were identified, of which 30 simple sequence repeat primer pairs were developed. Of these, 24 (80%) amplified polymerase chain reaction products of expected sizes. Twelve primer pairs amplified two to four bands, whereas another 12 primer pairs produced monomorphic banding patterns. Data for 12 loci were analyzed using POPGENE software version 1.32. A total of 29 alleles were produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers were used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines were clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of motif repeats in different alleles were major sources of polymorphisms.     

Development of silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) genetic maps using microsatellite and AFLP markers and a pseudo-testcross strategy

Silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are two of the four most important pond-cultured fish species inhabiting the major river basins of China. In the present study, genetic maps of silver carp and bighead carp were constructed using microsatellite and AFLP markers and a two-way pseudo-testcross strategy. To create the maps, 60 individuals were obtained from a cross of a single bighead carp (female) and a single silver carp (male). The silver carp map consisted of 271 markers (48 microsatellites and 223 AFLPs) that were assembled into 27 linkage groups, of which 22 contained at least four markers. The total length of the silver carp map was 952.2 cM, covering 82.8% of the estimated genome size. The bighead carp map consisted of 153 markers (27 microsatellites and 126 AFLPs) which were organized into 30 linkage groups, of which 19 contained at least four markers. The total length of the bighead carp map was 852.0 cM, covering 70.5% of the estimated genome size. Eighteen microsatellite markers were common to both maps. These maps will contribute to discovery of genes and genetic regions controlling traits in the two species of carp.     

A preliminary microsatellite genetic map of the ostrich (Struthio camelus)

Molecular genetic maps can provide information for the identification and localization of major genes associated with quantitative traits. However, there are currently no published genetic linkage maps for any ratites. Herein, a preliminary genetic map of ostrich was developed using a two-generation ostrich reference family by linkage analysis of 104 polymorphic microsatellite markers, including 40 novel markers reported in this study. A total of 35 microsatellite markers were placed into 13 linkage groups. Five linkage groups are composed of three or more loci, whereas the remaining eight groups each contained two markers. The sex-averaged map spans 365.4 cM. The marker interval of each linkage group ranges from 5.3 to 25.4 cM, and the average interval distance is 16.61 cM. The male map covers 342.7 cM, with an average intermarker distance of 15.58 cM, whereas the female map is 456.7 cM, with the average intermarker spacing of 20.76 cM. In order to screen the orthologous loci between ostrich and chicken, all of the flanking sequences of the 104 polymorphic loci, nine monomorphic loci and a further 12 reported microsatellite loci for ostrich were screened against the chicken genomic sequence using the BLAST algorithm (Altschul et al., 1990), and corresponding orthologs were found for 13 sequences. The microsatellite loci and genetic map developed in this study will be useful for QTL mapping, population genetics and phylogenetic studies in the ratite. In addition, the 13 orthologous loci identified in this study will be advantageous to the construction of a comparative genetic map between chicken and ostrich.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.