Microsatellite genotyping by primer extension and MALDI-ToF mass spectrometry

A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed. Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barleyMlo gene.     

Microsatellite standardization and evaluation of genotyping error in a large multi-partner research programme for conservation of Atlantic salmon (Salmo salar L.)

Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size ('size shifts'), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.     

Plant microsatellite genotyping with 4-color fluorescent detection using multiple-tailed primers

We extended the concept of fluorescent microsatellite genotyping with a single-universal tailed primer to the simultaneous use of three different tailed primers to allow multiplexed 4-color detection for medium throughput genotyping of plant species. The method was tested on Eucalyptus DNA samples using three forward primer sequences of human microsatellite markers labeled with different fluorescent dyes. The robustness of the method was tested for the simultaneous detection and genetic analysis of microsatellites in a genetic mapping experiment. This method allows reliable and cost-effective genotyping with the same level of multiplexing attained in regular microsatellite fluorescent detection assays. Besides the enhanced quality of the genotypic data provided by the fluorescent detection method when compared to colorimetric ones, the economy brought about by this method becomes greater with an increasing number of microsatellite markers. This method has been particularly useful for genotyping populations of several tropical tree species addressing community-wide population genetics and conservation questions.     

High Resolution Melting (HRM) analysis in eggplant (Solanum melongena L.): A tool for microsatellite genotyping and molecular characterization of a Greek Genebank collection

The conservation and characterization of eggplant (Solanum melongena L.) genetic resources in germplasm banks has been the basis of their use in breeding projects, which has resulted in the development of new cultivars. High Resolution Melting (HRM) analysis, combined with eight microsatellite markers, has been integrated in order to facilitate the molecular identification and characterization of the eggplant germplasm, collected from the National Genebank Collection of Greece. The eight microsatellite loci used were highly informative and generated sixty three HRM profiles, which were sufficient to discriminate all eggplant landraces and cultivars studied, highlighting its potential use for cultivar genotyping. The thirty six eggplant genotypes were classified into four clusters. Hence, this assay provided a fast, cost-effective and closed-tube microsatellite genotyping method, well suited for molecular characterization of eggplant cultivars.     

毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Availability of short microsatellite markers from butterfly museums and private specimens

Knowledge of the temporal changes in genetic diversity and structure is important for identifying factors causing a decline in threatened insect species, and for establishing conservation programs for these species. Thus, there is recently an increasing interest in the restoration of genetic diversity in conservation programs using DNA data from historical museum specimens. For butterfly specimens, we measured the yields and fragment sizes of the extracted DNA and investigated the genotyping success probability of nine short microsatellite markers (allele size 73–191 bp). We used leg samples of specimens of a medium‐sized butterfly species, Melitaea ambigua (Lepidoptera; Nymphalidae), collected from the 1960s to the 2010s. The yields of specimen‐extracted DNA longer than 150 bp decreased with increasing specimen age. There were negative correlations between the genotyping success probability and specimen age for each of all microsatellite markers. A negative correlation was also observed between the genotyping success probability and allele size of each microsatellite marker. We conclude that short microsatellite markers and analysis of recently obtained specimens are particularly suitable for microsatellite analysis of butterfly specimens.     

Microsatellite markers for population-genetic studies of shiitake (Lentinula edodes) strains

In order to analyze the genotyping of shiitake (Lentinula edodes, Berk.), one of commercially and widely grown edible mushrooms, we examined group of total 89 strains that are registered in Korea, Japan, and China respectively, using five microsatellite markers (Led A2, Led A8, Led B2, Led B6, and Led D6) registered in NCBI (National Center for Biotechnology Information). After we prepared synthesis of primers modified with 5′-FAM fluorescent dye and conducted PCR program, we obtained reasonable products. And then we performed microsatellite genotyping analysis using an ABI 3730xl Genetic Analyzer. According to genotyping analysis, the number of alleles of each microsatellite marker ranged from 5 to 14 with the average value of 8.2. The expected and the observed heterozygosity over all microsatellite ranged from 0.27–0.83 and 0.14–0.61. Among 5 microsatellite markers PIC (Polymorphism Information Content) values of Led A8, Led D6, and Led B6 resulted 0.82, 0.60, and 0.57 respectively. We observed that these values showed relatively high values discriminating from other values of microsatellite marker. The average of total values of Led A8, Led D6, and Led B6 came out 0.53 as result, which is higher than 0.5. As a result, this average can be subject to significant value to be used as marker. By using microsatellite marker we can analogically establish population relationships among shiitake (L. edodes) strains grown in East Asian region, develop new varieties, and propose discriminative criteria for different breeding and variety classification. Moreover, microsatellite markers enable us to obtain the genetic inheritance and information that can be used for protection of intellectual property rights.     

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.     

Ethnic-difference markers for use in mapping by admixture linkage disequilibrium

Mapping by admixture linkage disequilibrium (MALD) is a potentially powerful technique for the mapping of complex genetic diseases. The practical requirements of this method include (a) a set of markers spanning the genome that have large allele-frequency differences between the parental ethnicities contributing to the admixed population and (b) an understanding of the extent of admixture in the study population. To this end, a DNA-pooling technique was used to screen microsatellite and diallelic insertion/deletion markers for allele-frequency differences between putative representatives of the parental populations of the admixed Mexican American (MA) and African American (AA) populations. Markers with promising pooled differences were then confirmed by individual genotyping in both the parental and admixed populations. For the MA population, screening of >600 markers identified 151 ethnic-difference markers (EDMs) with delta>0.30 (where delta is the absolute value of each allele-frequency difference between two populations, summed over all marker alleles and divided by two) that are likely to be useful for MALD analysis. For the AA population, analysis of >400 markers identified 97 EDMs. In addition, individual genotyping of these markers in Pima Amerindians, Yavapai Amerindians, European American (EA) individuals, Africans from Zimbabwe, MA individuals, and AA individuals, as well as comparison to the CEPH genotyping set, suggests that the differences between subpopulations of an ethnicity are small for many markers with large interethnic differences. Estimates of admixture that are based on individual genotyping of these markers are consistent with a 60% EA:40% Amerindian contribution to MA populations and with a 20% EA:80% African contribution to AA populations. Taken together, these data suggest that EDMs with large interpopulation and small intrapopulation differences can be readily identified for MALD studies in both AA and MA populations.     

Non-destructive sampling of maternal DNA from the external shell of bird eggs

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.     

Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts

In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality samples was 96%, 24%, and 24% for microsatellite loci, and 98%, 97%, and 79% for SNPs, respectively. As measured by qPCR, good quality samples had a consistently high number of starting copies across all fragment sizes with little change between the smallest and largest size. In contrast, the intermediate and poor quality samples displayed decreases in starting copy number as fragment size increased, and was most pronounced with poor samples. Logistic regression of genotyping success by starting copy number indicated that in order to achieve at least 90% genotyping success, approximately 1,000 starting copies of nuclear DNA are necessary for microsatellite loci, and as few as 14 starting copies for SNP assays (but we recommend at least 50 copies to reduce genotyping error). While these guidelines apply specifically to Chinook salmon and the genetic markers included in this study, the principles are transferable to other species and markers due to the underlying process associated with template quantity and PCR amplification.     

Comparison of microsatellite and single nucleotide polymorphism markers for the genetic analysis of a Galloway cattle population

Highly informative genetic markers are essential for efficient management of cattle populations, as well as for food safety. After a decade of domination by microsatellite markers, a new type of genetic marker, single nucleotide polymorphism (SNP), has recently appeared on the scene. In the present study, the exclusion power of both kinds of markers with regards to individual identification and parental analysis was directly compared in a Galloway cattle population. Seventeen bovine microsatellites were distributed in three incremental marker sets (10, 14 and 17 microsatellite markers) and used for cattle genotyping. A set of 43 bovine SNP was used for genotyping the same cattle population. The accuracy of both kinds of markers in individual identification was evaluated using probability of identity estimations. These were 2.4 x 10(-8) for the 10 microsatellite set, 2.3 x 10(-11) for the 14 microsatellite set, and 1.4 x 10(-13) for the 17 microsatellite marker set. For the 43 SNP markers, the estimated probability of identity was 5.3 x 10(-11). The exclusion power of both kinds of markers in parental analysis was evaluated using paternity exclusion estimations, and, in addition to this, by estimation of the parental exclusion probability in 18 Galloway family trios. Paternity exclusion was estimated to be over 99% for microsatellites, and approx. 98% for SNP. Both, microsatellite and SNP sets of markers showed similar parental exclusion probabilities.     

Detection of a quantitative trait locus for both foliage and tuber resistance to late blight [Phytophthora infestans (Mont.) de Bary] on chromosome 4 of a dihaploid potato clone (Solanum tuberosum subsp. tuberosum)

Linkage analysis, Kruskal–Wallis analysis, interval mapping and graphical genotyping were performed on a potato diploid backcross family comprising 120 clones segregating for resistance to late blight. A hybrid between the Solanum tuberosum dihaploid clone PDH247 and the long-day-adapted S. phureja clone DB226(70) had been crossed to DB226(70) to produce the backcross family. Eighteen AFLP primer combinations provided 186 and 123 informative maternal and paternal markers respectively, with 63 markers in common to both parents. Eleven microsatellite (SSR) markers proved useful for identifying chromosomes. Linkage maps of both backcross parents were constructed. The results of a Kruskal–Wallis analysis, interval mapping and graphical genotyping were all consistent with a QTL or QTLs for blight resistance between two AFLP markers 30 cM apart on chromosome 4, which was identified by a microsatellite marker. The simplest explanation of the results is a single QTL with an allele from the dihaploid parent conferring resistance to race 1, 4 of P. infestans in the foliage in the glasshouse and to race 1, 2, 3, 4, 6, 7 in the foliage in the field and in tubers from glasshouse raised plants. The QTL was of large effect, and explained 78 and 51% of the variation in phenotypic scores for foliage blight in the glasshouse and field respectively, as well as 27% of the variation in tuber blight. Graphical genotyping and the differences in blight scores between the parental clones showed that all of the foliage blight resistance is accounted for by chromosome 4, whereas undetected QTLs for tuber resistance probably exist on other chromosomes. Graphical genotyping also explained the lack of precision in mapping the QTL(s) in terms of lack of appropriate recombinant chromosomes.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.     

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.     

鳙基于10个微卫星标记的亲子鉴定分析

为开展鳙(Hypophthalmichthys nobilis)家系选育工作,本研究进行了基于微卫星标记的亲子鉴定研究。试验中筛选了10个扩增效率较高的微卫星标记,通过引物荧光修饰,引物结合毛细管电泳分型技术,对鳙48尾亲本及384尾子代进行了基因分型,并计算了等位基因频率和模拟分析和亲子鉴定等分析。结果发现,各位点的等位基因数介于4~13之间,其中9个位点均具有较高的多态性和杂合度(PIC>0.5,He>0.5),研究发现位点的多态性信息含量(PIC)与亲本对排除率(E-PP)存在显著正相关(p<0.01)。模拟分析结果显示,该10个标记预计可用于已知性别的50组亲本(100尾)或未知性别的50尾亲本的鉴定分析(鉴别成功率>95%)。亲子鉴定发现,对试验中2个交配组(每组12对亲本)的鉴别成功率分别为98.96%和100%;且父母本对子代的贡献率存在极显著差异(p<0.01)。通过累积位点的鉴定分析发现,当标记数为7个和9个时分别能满足试验中12组和24组亲本对应子代的鉴定分析(鉴别率>95%),模拟分析和亲子鉴定分析成功率趋势基本符合。本研究所开发的亲子鉴定技术可为鳙家系选育提供技术支持。     

High resolution melting analysis of almond SNPs derived from ESTs

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.     

Analysis of microsatellite loci in tree of heaven (<Emphasis Type="Italic">Ailanthus altissima</Emphasis> (Mill.) Swingle) using SSR-GBS

Microsatellite markers are still the marker of choice for many research questions in the field of forest genetics. However, the number of available markers is often low for species that have not been studied intensively like the tree of heaven (Ailanthus altissima). During the last decade, next-generation sequencing (NGS) has offered advanced techniques for efficiently identifying microsatellite markers and accurately genotyping samples. Here, we identify new microsatellite markers for the tree of heaven by applying an NGS-based method using the Illumina MiSeq platform. NGS technology was proved to be an effective method for fast and cost-efficient identification of microsatellite markers by implementing a genotyping-by-sequencing approach based on Illumina amplicon sequencing (SSR-GBS). We screened three populations from Eastern Austria for genetic variation at 19 newly identified microsatellite loci. We tested two different genotyping approaches: (1) considering only allele lengths (forming a so-called “allele length dataset”), (2) taking also single nucleotide polymorphisms (SNPs) within the amplified fragments into account (forming a so-called “SNP dataset”). The results revealed higher values for all genetic diversity parameters, as well as a better resolution of genetic assignment, when the latter approach was followed. Thus, by taking advantage of sequence information which is provided by SSR-GBS, one may achieve considerable gains in performance using the same marker set. The developed markers provide a cost-efficient tool for genotyping populations of tree of heaven and the approach presented here promises to be of high value for medium throughput genotyping applications in non-model forest tree species. We will use this method to widen the perspectives for further population genetic investigations of the tree of heaven.