megasat: automated inference of microsatellite genotypes from sequence data

megasat is software that enables genotyping of microsatellite loci using next‐generation sequencing data. Microsatellites are amplified in large multiplexes, and then sequenced in pooled amplicons. megasat reads sequence files and automatically scores microsatellite genotypes. It uses fuzzy matches to allow for sequencing errors and applies decision rules to account for amplification artefacts, including nontarget amplification products, replication slippage during PCR (amplification stutter) and differential amplification of alleles. An important feature of megasat is the generation of histograms of the length–frequency distributions of amplification products for each locus and each individual. These histograms, analogous to electropherograms traditionally used to score microsatellite genotypes, enable rapid evaluation and editing of automatically scored genotypes. megasat is written in Perl, runs on Windows, Mac OS X and Linux systems, and includes a simple graphical user interface. We demonstrate megasat using data from guppy, Poecilia reticulata. We genotype 1024 guppies at 43 microsatellites per run on an Illumina MiSeq sequencer. We evaluated the accuracy of automatically called genotypes using two methods, based on pedigree and repeat genotyping data, and obtained estimates of mean genotyping error rates of 0.021 and 0.012. In both estimates, three loci accounted for a disproportionate fraction of genotyping errors; conversely, 26 loci were scored with 0–1 detected error (error rate ≤0.007). Our results show that with appropriate selection of loci, automated genotyping of microsatellite loci can be achieved with very high throughput, low genotyping error and very low genotyping costs.     

Water-soluble proteins of mature barley endosperm: genetic control,polymorphism, and linkage with β-amylase and spring/winter habit

Summary Water-soluble proteins (WSP-2 and WSP-3) and -amylase (-AMY-1) were extracted from mature endosperms of 44 spring and 39 winter barley genotypes. The protein and enzyme isoforms were separated in isoelectric focusing gels with a pH gradient of 4–6.5. The Wsp-3 and -Amy-1 loci were located to chromosomes 4H using the wheat/barley chromosome addition lines. Segregation analysis of F₂ and doubled haploid populations showed Wsp-2 and -Amy-1 to be tightly linked, with a map distance of 11 cMorgans. Isoforms of WSP-2 possessed similar pIs to that of WSP-3 and overlapping bands were observed in the gels. These bands segregated independently in F₂ and doubled haploid populations, implying two unlinked genes. All three loci were found to be polymorphic: two alleles were detected at the Wsp-2 locus, three at Wsp-3 and two at -Amy-1. The frequency of alleles at all three loci was found to be different in winter and spring genotypes. Spring genotypes possessed a wider range of phenotypes than winter genotypes. Spring and winter genotypes could be distinguished on the basis of WSP-3 and - AMY-1 phenotypes. The linkage between Wsp-3 and -Amy-1 loci and genes controlling spring/winter habit on chromosome 4H is discussed. It is concluded that Wsp-3 and -Amy-1 can be used as genetic markers for spring/winter habit in barley genetic research and breeding.     

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.     

Isolation and characterization of microsatellite DNA loci in Aquilegia sp.

We obtained a microsatellite‐enriched genomic library isolated from the tissue of a single columbine (Aquilegia sp.) plant taken from a southwestern USA natural population. The primers developed for these microsatellite loci performed consistently in polymerase chain reactions and yielded multiallelic genotypes with relatively high observed heterozygosities. We describe polymerase chain reaction primers and conditions to amplify 16 unique, codominant di‐, tri‐ and tetra‐nucleotide microsatellite DNA loci so that other population biology researchers using columbine natural populations as a model system may benefit.     

Assignment of allelic configuration in polyploids using the MAC-PR (microsatellite DNA allele counting—peak ratios) method

Polysomic inheritance frequently results in the simultaneous occurrence of several microsatellite DNA alleles on a single locus. The MAC-PR (microsatellite DNA allele counting—peak ratios) method was recently developed for the analysis of polyploid plants and makes use of the quantitative values for microsatellite allele peak areas. To date, this approach has only been used in plants with known genetic relationships. We report here the application of MAC-PR for the first time to random samples of unknown pedigrees. We analysed six microsatellite loci using a set of tetraploid ornamental rose (Rosa × hybrida L.) varieties. For each locus, all alleles were analysed in pairwise combinations in order to determine their copy number in the individual samples. This was accomplished by calculating the ratios between the peak areas for two alleles in all of the samples where these two alleles occurred together. The allele peak ratios observed were plotted in a histogram, and those histograms that produced at least two well-separated groups were selected for further analysis. Mean allelic peak ratio values for these groups were compared to the relationships expected between alleles in hypothetical configurations of the locus investigated. Using this approach, we were able to assign precise allelic configurations (the actual genotype) to almost all of the varieties analysed for five of the six loci investigated. MAC-PR also appears to be a very effective tool for detecting null alleles in polyploid species.Communicated by C. Möllers     

A somaclonal variant of wheat with additional β-amylase isozymes

Summary The progeny of 149 plants regenerated from tissue culture of immature wheat (Triticum aestivum) embryos were screened for variation in their grain -amylase isozyme pattern. One regenerant was found which was heterozygous for a variant pattern characterized by the presence of at least five new isozyme bands, as well as an increased intensity in existing bands in two more positions. The F₂ of a homozygous variant crossed back to the parent segregated in an approximate 31 ratio but resolution of the gels was not sufficient to distinguish whether this represents a dominant or co-dominant single mutant gene. No chromosome abnormalities were evident in mitosis or meiosis of the homozygous variant or in the F₁ of the variant crossed back to the parent. No recombination has been seen between the variant bands and production of multiple bands from a single locus is consistent with the nature of the known -amylase loci. However, the variant bands were not evident in a survey of 111 diverse genotypes, nor were they present in developing grain of the parent cultivar. Therefore, this variant could represent a rare mutation leading to expression of a currently unexpressed locus.     

Development of a cost-effective high-throughput process of microsatellite analysis involving miniaturized multiplexed PCR amplification and automated allele identification

Background

Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing.

Results

To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software.

Conclusions

In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.

     

Genetics of seed storage proteins in the love tree Cercis siliquastrum L. (Fabaceae)

The patterns of C. siliquastrum seed storage proteins (cercins) are described using SDS-polyacrylamide gel electrophoresis. The polypeptides detected had very different molecular weights (ranging from 168 to 34 KDa) which, together with their high homogeneity, produced a very good resolution of bands. These proteins could be ascribed to five different loci. The analysis of seed sets of individual trees indicated that the love tree is almost completely autogamous with less than 5% of outcrosses. Although this mode of reproduction seems to produce a decrease in heterozygote frequency among the seeds of the population analysed, the levels of variability detected were very high for an autogamous plant: all of the loci were polymorphic, with a mean heterozygosity of 0.327 and a polymorphic index of 0.412. Protein segregation revealed a strong genetic linkage between three cercin loci (a, c and d) while the other two are independent.     

An improved high yield method to obtain microsatellite genotypes from red deer antlers up to 200 years old

Analysis of DNA from older samples, such as museum specimen, is a promising approach to studying genetics of populations and ecological processes across several generations. Here, we present a method for extracting high quality nuclear DNA for microsatellite analysis from antlers of red deer (Cervus elaphus). The genotyping of individuals was based on nine microsatellite loci. Because the amount of DNA found in antlers was high, we could reduce the amount of sample and chemicals used and shorten the decalcification time in comparison to other methods. Using these methods, we obtained genotypes from antlers up to 200 years old.     

Tightly linked di- and tri-nucleotide microsatellites do not evolve in complete independence: evidence from linked (TA)n and (TAA)n microsatellites of chickpea (Cicer arietinum L.)

In order to understand the dynamics of microsatellite evolution, we have studied allelic variation at a closely linked (TA) _n and (TAA) _n microsatellite loci in 114 land races of chickpea (Cicer arietinum L.), sampled worldwide. These two loci are separated by 27 bp. The two loci showed a very high degree of polymorphism and hence the combined length with the genetic diversity of 0.93, 0.90 and 0.98 for (TAA) _n , (TA) _n and the combined length, respectively. Using the variation data at the linked loci, a standardized index of linkage disequilibrium was also computed (I ^S _A =0.092), which tests the null hypothesis of no linkage and was significant, indicating the presence of linkage disequilibrium. Furthermore, the dynamics of allelic variation showed that there is a threshold combined length, below which both (TAA) _n and (TA) _n loci evolve independently, and above which, if one locus increase in size, the other closely linked locus has a tendency to decrease its size and vice versa, without change in the overall ratio of (TAA) _n and (TA) _n allele sizes at the region. This result indicates that there are processes in the cell, which read the combined size of the two loci both for proportion and length and determine the direction of tightly linked di- and tri-nucleotide repeat evolution.Communicated by R. Hagemann     

Best Practices and Joint Calling of the HumanExome BeadChip: The CHARGE Consortium

Genotyping arrays are a cost effective approach when typing previously-identified genetic polymorphisms in large numbers of samples. One limitation of genotyping arrays with rare variants (e.g., minor allele frequency [MAF] <0.01) is the difficulty that automated clustering algorithms have to accurately detect and assign genotype calls. Combining intensity data from large numbers of samples may increase the ability to accurately call the genotypes of rare variants. Approximately 62,000 ethnically diverse samples from eleven Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium cohorts were genotyped with the Illumina HumanExome BeadChip across seven genotyping centers. The raw data files for the samples were assembled into a single project for joint calling. To assess the quality of the joint calling, concordance of genotypes in a subset of individuals having both exome chip and exome sequence data was analyzed. After exclusion of low performing SNPs on the exome chip and non-overlap of SNPs derived from sequence data, genotypes of 185,119 variants (11,356 were monomorphic) were compared in 530 individuals that had whole exome sequence data. A total of 98,113,070 pairs of genotypes were tested and 99.77% were concordant, 0.14% had missing data, and 0.09% were discordant. We report that joint calling allows the ability to accurately genotype rare variation using array technology when large sample sizes are available and best practices are followed. The cluster file from this experiment is available at www.chargeconsortium.com/main/exomechip.     

Genetic Analysis of Selected Strains of Eastern Oyster (Crassostrea virginica Gmelin) Using AFLP and Microsatellite Markers

Amplified fragment length polymorphisms (AFLPs) and microsatellite markers were used to examine genetic variation and divergence in 4 selected strains (DBH, NEH, FMF, and CTS) and 1 wild population (DBW) of the eastern oyster Crassostrea virginica Gmelin. Eighty-six AFLP markers (from 3 primer pairs) and 5 microsatellite loci were used for the analysis of 30 oysters from each of the 5 populations. Microsatellite loci were considerably more variable than AFLPs. The observed heterozygosity ranged from 0.560 to 0.640 across populations for microsatellites, and from 0.186 to 0.207 for AFLPs. Both F_st and _PT of microsatellite data and _PT statistics of AFLP data revealed significant divergence between all pairs of populations. There was no significant reduction in heterozygosity in all 4 selected strains; however, the number of alleles per locus was considerably lower in the selected strains than in the wild population. Two strains subjected to long-term selection for disease resistance shared frequency shifts at a few loci, which deserve further analysis to determine if they are linked to disease-resistance genes.     

Establishment of molecular markers and linkage groups in two F2 populations of Upland cotton

Two F₂ populations of cotton (Gossypium hirsutum L.) from the crosses of HS46 x MARCABUCAG8US-1-88 (MAR) and HS46 x Pee Dee 5363 (PD5363) were characterized for restriction fragment length polymorphisms (RFLPs) using DNA probes. Seventy-three probe/enzyme combinations were used in the HS46 x MAR population analysis, which resulted in 42 informative polymorphic fragments. These 42 moleclar markers represented 26 polymorphic loci, which consisted of 15 codominant and 11 dominant (+/-) genotypes. Chi-square analyses of these loci fit expected genotypic ratios of 121 and 31, respectively An analysis of these loci with the MAPMAKER program resulted in the establishment of four linkage groups A, B, C, and D with 4,2,2, and 2 loci, respectively, as well as 16 unlinked loci. Six probe-enzyme combinations were assayed on the HS46 x PD5363 population, which resulted in 11 informative polymorphic fragments. These 11 fragments represented 6 polymorphic loci, 1 dominant (+/-) and 5 codominant genotypes. The MAPMAKER analysis of these loci yielded 2 linked loci. Thus, a total of 53 polymorphic fragments and 32 polymorphic loci, representing five linkage groups, were identified among the two families.Contribution of the USDA-ARS in cooperation with the Miss Agric For Exp Stn.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

SNiPer-HD: improved genotype calling accuracy by an expectation-maximization algorithm for high-density SNP arrays

MOTIVATION: The technology to genotype single nucleotide polymorphisms (SNPs) at extremely high densities provides for hypothesis-free genome-wide scans for common polymorphisms associated with complex disease. However, we find that some errors introduced by commonly employed genotyping algorithms may lead to inflation of false associations between markers and phenotype. RESULTS: We have developed a novel SNP genotype calling program, SNiPer-High Density (SNiPer-HD), for highly accurate genotype calling across hundreds of thousands of SNPs. The program employs an expectation-maximization (EM) algorithm with parameters based on a training sample set. The algorithm choice allows for highly accurate genotyping for most SNPs. Also, we introduce a quality control metric for each assayed SNP, such that poor-behaving SNPs can be filtered using a metric correlating to genotype class separation in the calling algorithm. SNiPer-HD is superior to the standard dynamic modeling algorithm and is complementary and non-redundant to other algorithms, such as BRLMM. Implementing multiple algorithms together may provide highly accurate genotyping calls, without inflation of false positives due to systematically miss-called SNPs. A reliable and accurate set of SNP genotypes for increasingly dense panels will eliminate some false association signals and false negative signals, allowing for rapid identification of disease susceptibility loci for complex traits. AVAILABILITY: SNiPer-HD is available at TGen's website: http://www.tgen.org/neurogenomics/data.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

Analysis of a marsupial MHC region containing two recently duplicated class I loci

A 37-kb cosmid containing two complete major histocompatibility complex (MHC) class I chain loci from the opossum Monodelphis domestica was isolated, fully sequenced, and characterized. This sequence represents the largest contiguous genomic sequence reported for the MHC region of a nonplacental mammal. Based on particular conserved amino acid residues, and limited expression analyses, the two MHC-I loci, designated ModoUB and ModoUC, appear to encode functional MHC-I molecules. The two coding regions are 98% identical at the nucleotide level; however, their promoter regions differ significantly. Two CpG islands present in the cosmid sequence correspond to the two coding regions. Twelve microsatellites and six retroelements were also present in the cosmid. The retroelements share highest sequence homology to the CORE–SINE family of retroelements. Due to high sequence identity, it is very likely that ModoUB and ModoUC loci are products of recent gene duplication that occurred less than 4 million years ago.     

First microsatellite markers for <Emphasis Type="Italic">Paspalum plicatulum</Emphasis> (Poaceae) characterization and cross-amplification in different <Emphasis Type="Italic">Paspalum</Emphasis> species of the Plicatula group

Background

Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. It is a member of Plicatula, which has historically been recognized as a highly complex group containing species of ecological, ornamental and forage importance. The complex nature of the P. plicatulum genome makes it a challenging species for genetic research. This study aimed to develop and characterize microsatellite molecular markers in P. plicatulum and to evaluate their transferability to other Plicatula group species.

Findings

Microsatellite sequences were identified from three enriched libraries from P. plicatulum. Specific primers were designed, and 25 displayed polymorphism when screened across 48 polyploid Paspalum spp. genotypes. The number of bands per locus ranged from 2 to 17, with a mean of 8.65. Private bands for each species were identified; the highest number of private bands was observed for P. plicatulum in 52% of the loci analyzed. The mean polymorphism information content of all loci was 0.69, and the mean discriminatory power was 0.82. Microsatellite markers were satisfactorily cross-amplified for the eight tested Plicatula-group Paspalum species, with P. atratum exhibiting the highest transferability rate (89.86%). STRUCTURE and Discriminant Analysis of Principal Components separated accessions into three groups but did not reveal separation of the accessions according to species.

Conclusions

This study describes the first microsatellite markers in P. plicatulum, which are polymorphic, efficient for the detection and quantification of genetic variation, and show high transferability into other species of the Plicatula group. This set of markers can be used in future genetic and molecular studies necessary for the proper development of conservation and breeding programs. Private bands within the markers can be used to assist in species identification.