双色荧光杂交芯片在近交系小鼠遗传监测中的应用

应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术进行近交系小鼠遗传监测。应用双色荧光杂交芯片技术对4个品系近交系小鼠的多个基因组DNA样本进行SNP分型,整合6个SNP位点的芯片杂交信息,对样本所属品系进行判断。研究结果表明SNP检测方法-双色荧光杂交芯片技术能够对选定的6个SNP位点进行高准确率分型;双色荧光杂交芯片技术是一种高通量SNP检测的良好工具,适合于对少量近交系品系来源的大样本量小鼠进行遗传污染监测和品系鉴定,并具有扩大应用的潜力。     

SNP genotyping for the genetic monitoring of laboratory mice by using a microarray-based method with dualcolour fluorescence hybridisation

Ensuring the genetic homogeneity of the mice used in laboratory experiments contributes to the Reduction aspect of the Three Rs, by maximising the quality of the data obtained from any animals that are used for these purposes, and ultimately reducing the numbers of animals used. Single nucleotide polymorphism (SNP) genotyping is especially suitable for use in the analysis of the genetic purity of model organisms such as the mouse, because bi-allelic markers remain fully informative when used to characterise crosses between inbred strains. Here, we attempted to apply a microarray-based method for a SNP marker to monitor the genetic quality of inbred mouse strains, so as to validate the reliability, stability and applicability of this SNP genotyping panel. The amplified PCR products containing four different SNP loci from four inbred mouse strains were spotted and immobilised onto amino-modified glass slides to generate a microarray. This was then interrogated through hybridisation with dual-colour probes, to determine the SNP genotypes of each sample. The results indicated that this microarray-based method could effectively determine the genotypes of the four selected SNPs with a high degree of accuracy. We have developed a new SNP genotyping technique for effective use in the genetic monitoring of inbred mouse strains.     

Identification of a genetic contamination in a commercial mouse strain using two panels of polymorphic markers

Rapid detection of genetic contamination is critical in mouse studies involving inbred strains. During a Quantitative Trait Locus (QTL) study using simple sequence length polymorphism (SSLP) markers, we noticed heterozygosity at some loci of a commercially available inbred C57BL/6N mouse strain, suggesting a contamination by another mouse strain. A panel of 100 single-nucleotide polymorphism (SNP) markers was used to confirm and specify the genetic contamination suspected. Retrospective analyses demonstrated that the contamination took place as early as autumn 2003 and has persisted ever since at a fairly constant level. Contaminating alleles most probably originated from a DBA strain. Our data demonstrate the suitability of SNP markers for rapid detection and identification of the source of genetic contamination. Further, our results show the importance of a state-of-the-art genetic monitoring of the authenticity of murine inbred strains.     

A comprehensive SNP-based genetic analysis of inbred mouse strains

Dense genetic maps of mammalian genomes facilitate a variety of biological studies including the mapping of polygenic traits, positional cloning of monogenic traits, mapping of quantitative or qualitative trait loci, marker association, allelic imbalance, speed congenic construction, and evolutionary or phylogenetic comparison. In particular, single nucleotide polymorphisms (SNPs) have proved useful because of their abundance and compatibility with multiple high-throughput technology platforms. SNP genotyping is especially suited for the genetic analysis of model organisms such as the mouse because biallelic markers remain fully informative when used to characterize crosses between inbred strains. Here we report the mapping and genotyping of 673 SNPs (including 519 novel SNPs) in 55 of the most commonly used mouse strains. These data have allowed us to construct a phylogenetic tree that correlates and expands known genealogical relationships and clarifies the origin of strains previously having an uncertain ancestry. All 55 inbred strains are distinguishable genetically using this SNP panel. Our data reveal an uneven SNP distribution consistent with a mosaic pattern of inheritance and provide some insight into the changing dynamics of the physical architecture of the genome. Furthermore, these data represent a valuable resource for the selection of markers and the design of experiments that require the genetic distinction of any pair of mouse inbred strains such as the generation of congenic mice, positional cloning, and the mapping of quantitative or qualitative trait loci.The content of this publication does not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.     

MSQT for choosing SNP assays from multiple DNA alignments

MOTIVATION: One challenging aspect of genotyping and association mapping projects is often the identification of markers that are informative between groups of individuals and to convert these into genotyping assays. RESULTS: The Multiple SNP Query Tool (MSQT) extracts SNP information from multiple sequence alignments, stores it in a database, provides a web interface to query the database and outputs SNP information in a format directly applicable for SNP-assay design. MSQT was applied to Arabidopsis thaliana sequence data to develop SNP genotyping assays that distinguish a recurrent parent (Col-0) from five other strains. SNPs with intermediate allele frequencies were also identified and developed into markers suitable for efficient genetic mapping among random pairs of wild strains. AVAILABILITY: The source code for MSQT is available at http://msqt.weigelworld.org, together with an online instance of MSQT containing data on 1214 sequenced fragments from 96 ecotypes (wild inbred strains) of the reference plant A. thaliana. All SNP genotyping assays are available in several formats for broad community use. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

基于PCR-LDR平台的近交系小鼠遗传质量快速检测方法

目的建立基于PCR-LDR平台的近交系小鼠SNP快速分型方法,用于检测实验小鼠的遗传质量与品系纯度。方法利用可移植性极高的PCR-LDR技术,以常见近交系小鼠为研究对象,选取了21条染色体上的45个SNP位点,分别设计引物和探针,经过筛选和验证,建立了多重PCR-LDR(polymerase chain reaction and ligase detection reaction,PCR-LDR)分型方案。结果四组多重PCR-LDR可实现45个SNP位点的基因分型,其中43个、44个与45个SNP在样本中的检出率分别为100%、90.9%与36.4%。所有样本经分型确定为纯合体,并得到了常见近交系小鼠SNP位点信息。结论实现了常见近交系小鼠快速、高通量的基因分型,可用于遗传质量检测和品系鉴定。     

Characterization of 151 SNPs for population structure analysis of the endangered Tatra chamois (Rupicapra rupicapra tatrica) and its relative, the Alpine chamois (R. r. rupicapra)

We present 151 SNPs (single nucleotide polymorphisms) identified in Tatra and Alpine chamois individuals after genotyping with the Bovine SNP50 Genotyping BeadChip (Illumina^®). Population structure analysis based on the identified 151 SNPs as well as a subset of 48 SNPs were able to give information about geographic origin of each individual making the markers suitable for future evaluation and monitoring of the genetic status of the Tatra and Alpine chamois. Our study demonstrates how it is possible to quickly identify informative SNPs in non-model organisms based on a SNP marker panel created for a related domestic species.     

High-throughput SNP genotyping with the GoldenGate assay in maize

Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the genomes of most plant species. They have become an ideal marker system for genetic research in many crops. Several high throughput platforms have been developed that allow rapid and simultaneous genotyping of up to a million SNP markers. In this study, a custom GoldenGate assay containing 1,536 SNPs was developed based on public SNP information for maize and used to genotype two recombinant inbred line (RIL) populations (Zong3 x 87-1, and B73 x By804) and a panel of 154 diverse inbred lines. Over 90% of the SNPs were successfully scored in the diversity panel and the two RIL populations, with a genotyping error rate of less than 2%. A total of 975 SNP markers detected polymorphism in at least one of the two mapping populations, with a polymorphic rate of 38.5% in Zong3 x 87-1 and 52.6% in B73 x By804. The polymorphic SNPs in B73 x By804 have been integrated with previously mapped simple sequence repeat markers to construct a high-density linkage map containing 662 markers with a total length of 1,673.7 cM and an average of 2.53 cM between two markers. The minor allelic frequency (MAF) was distributed evenly across 10 continued classes from 0.05 to 0.5, and about 16% of the SNP markers had a MAF below 10% in the diversity panel. Polymorphism rates for individual SNP markers in pair-wise comparisons of genotypes tested ranged from 0.3 to 63.8% with an average of 36.3%. Most SNPs used in this GoldenGate assay appear to be equally useful for diversity analysis, marker-trait association studies, and marker-aided breeding.     

Single nucleotide polymorphisms for pig identification and parentage exclusion

Single nucleotide polymorphisms (SNPs) have become an important type of marker for commercial diagnostic and parentage genotyping applications as automated genotyping systems have been developed that yield accurate genotypes. Unfortunately, allele frequencies for public SNP markers in commercial pig populations have not been available. To fulfil this need, SNP markers previously mapped in the USMARC swine reference population were tested in a panel of 155 boars that were representative of US purebred Duroc, Hampshire, Landrace and Yorkshire populations. Multiplex assay groups of 5-7 SNP assays/group were designed and genotypes were determined using Sequenom's massarray system. Of 80 SNPs that were evaluated, 60 SNPs with minor allele frequencies >0.15 were selected for the final panel of markers. Overall identity power across breeds was 4.6 x 10(-23), but within-breed values ranged from 4.3 x 10(-14) (Hampshire) to 2.6 x 10(-22) (Yorkshire). Parentage exclusion probability with only one sampled parent was 0.9974 (all data) and ranged from 0.9594 (Hampshire) to 0.9963 (Yorkshire) within breeds. Sire exclusion probability when the dam's genotype was known was 0.99998 (all data) and ranged from 0.99868 (Hampshire) to 0.99997 (Yorkshire) within breeds. Power of exclusion was compared between the 60 SNP and 10 microsatellite markers. The parental exclusion probabilities for SNP and microsatellite marker panels were similar, but the SNP panel was much more sensitive for individual identification. This panel of SNP markers is theoretically sufficient for individual identification of any pig in the world and is publicly available.     

Development of SNP markers for C57BL/6N-derived mouse inbred strains

C57BL/6N inbred mice are used as the genetic background for producing knockout mice in large-scale projects worldwide; however, the genetic divergence among C57BL/6N-derived substrains has not been verified. Here, we identified novel single nucleotide polymorphisms (SNPs) specific to the C57BL/6NJ strain and selected useful SNPs for the genetic monitoring of C57BL/6N-derived substrains. Informative SNPs were selected from the public SNP database at the Wellcome Trust Sanger Institute by comparing sequence data from C57BL/6NJ and C57BL/6J mice. A total of 1,361 candidate SNPs from the SNP database could distinguish the C57BL/6NJ strain from 12 other inbred strains. We confirmed 277 C57BL/6NJ-specific SNPs including 10 nonsynonymous SNPs by direct sequencing, and selected 100 useful SNPs that cover all of the chromosomes except Y. Genotyping of 11 C57BL/6N-derived substrains at these 100 SNP loci demonstrated genetic differences among the substrains. This information will be useful for accurate genetic monitoring of mouse strains with a C57BL/6N-derived background.     

High-Throughput RAD-SNP Genotyping for Characterization of Sugar Beet Genotypes

High-throughput single-nucleotide polymorphism (SNP) genotyping provides a rapid way of developing resourceful sets of markers for delineating genetic structure and for understanding the basis of the taxonomic discrimination. In this paper, we present a panel of 192 SNPs for effective genotyping in sugar beet using a high-throughput marker array technology, QuantStudio 12K Flex system, coupled with Taqman OpenArray technology. The selected SNPs were evaluated for genetic diversity among a set of 150 individuals representing 15 genotypes (10 individuals each) from five cytoplasmic male steriles (CMSs), five pollinators, and five commercial varieties. We demonstrated that the proposed panel of 192 SNPs effectively differentiated the studied genotypes. A higher degree of polymorphism was observed among the CMSs as compared to pollinators and commercial varieties. PCoA and STRUCTURE analysis revealed that CMSs, pollinators, and varieties clustered into three distinct subpopulations. Our results demonstrate the utility of the identified panel of 192 SNPs coupled with TaqMan OpenArray technology as a wide set of markers for high-throughput SNP genotyping in sugar beet.     

High-resolution, high-throughput SNP mapping in Drosophila melanogaster

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.     

1 + 1 = 3: Development and Validation of a SNP-Based Algorithm to Identify Genetic Contributions from Three Distinct Inbred Mouse Strains

State-of-the-art, genome-wide assessment of mouse genetic background uses single nucleotide polymorphism (SNP) PCR. As SNP analysis can use multiplex testing, it is amenable to high-throughput analysis and is the preferred method for shared resource facilities that offer genetic background assessment of mouse genomes. However, a typical individual SNP query yields only two alleles (A vs. B), limiting the application of this methodology to distinguishing contributions from no more than two inbred mouse strains. By contrast, simple sequence length polymorphism (SSLP) analysis yields multiple alleles but is not amenable to high-throughput testing. We sought to devise a SNP-based technique to identify donor strain origins when three distinct mouse strains potentially contribute to the genetic makeup of an individual mouse. A computational approach was used to devise a three-strain analysis (3SA) algorithm that would permit identification of three genetic backgrounds while still using a binary-output SNP platform. A panel of 15 mosaic mice with contributions from BALB/c, C57Bl/6, and DBA/2 genetic backgrounds was bred and analyzed using a genome-wide SNP panel using 1449 markers. The 3SA algorithm was applied and then validated using SSLP. The 3SA algorithm assigned 85% of 1449 SNPs as informative for the C57Bl/6, BALB/c, or DBA/2 backgrounds, respectively. Testing the panel of 15 F2 mice, the 3SA algorithm predicted donor strain origins genome-wide. Donor strain origins predicted by the 3SA algorithm correlated perfectly with results from individual SSLP markers located on five different chromosomes (n=70 tests). We have established and validated an analysis algorithm based on binary SNP data that can successfully identify the donor strain origins of chromosomal regions in mice that are bred from three distinct inbred mouse strains.     

Comparison of SSRs and SNPs in assessment of genetic relatedness in maize

Advances in high-throughput SNP genotyping and genome sequencing technologies have enabled genome-wide association mapping in dissecting the genetic basis of complex quantitative traits. In this study, 82 SSRs and 884 SNPs with minor allele frequencies (MAF) over 0.20 were used to compare their ability to assess population structure, principal component analysis (PCA) and relative kinship in a maize association panel consisting of 154 inbred lines. Compared to SNPs, SSRs provided more information on genetic diversity. The expected heterozygosity (He) of SSRs and SNPs averaged 0.65 and 0.44, and the polymorphic information content of these two markers was 0.61 and 0.34 in this panel, respectively. Additionally, SSRs performed better at clustering all lines into groups using STRUCTURE and PCA approaches, and estimating relative kinship. For both marker systems, the same clusters were observed based on PCA and the first two eigenvectors accounted for similar percentage of genetic variations in this panel. The correlation coefficients of each eigenvector from SSRs and SNPs decreased sharply when the eigenvector varied from 1 to 3, but kept around 0 when the eigenvector were over 3. The kinship estimates based on SSRs and SNPs were moderately correlated (r (2)?=?0.69). All these results suggest that SSR markers with moderate density are more informative than SNPs for assessing genetic relatedness in maize association mapping panels.     

SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napus

Oilseed rape (Brassica napus) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome). The presence of these two genomes makes single nucleotide polymorphism (SNP) marker identification and SNP analysis more challenging than in diploid species, as for a given locus usually two versions of a DNA sequence (based on the two ancestral genomes) have to be analyzed simultaneously during SNP identification and analysis. One hundred amplicons derived from expressed sequence tag (ESTs) were analyzed to identify SNPs in a panel of oilseed rape varieties and within two sister species representing the ancestral genomes. A total of 604 SNPs were identified, averaging one SNP in every 42 bp. It was possible to clearly discriminate SNPs that are polymorphic between different plant varieties from SNPs differentiating the two ancestral genomes. To validate the identified SNPs for their use in genetic analysis, we have developed Illumina GoldenGate assays for some of the identified SNPs. Through the analysis of a number of oilseed rape varieties and mapping populations with GoldenGate assays, we were able to identify a number of different segregation patterns in allotetraploid oilseed rape. The majority of the identified SNP markers can be readily used for genetic mapping, showing that amplicon sequencing and Illumina GoldenGate assays can be used to reliably identify SNP markers in tetraploid oilseed rape and to convert them into successful SNP assays that can be used for genetic analysis.     

Comparison of four flow cytometric SNP detection assays and their use in plant improvement

Single nucleotide polymorphisms (SNPs) are attractive DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed, but it is unclear which assays are best suited and most efficient for various types of plant improvement research. The objective of this study was to compare the accuracy, efficiency, and cost of four SNP genotyping assays: single-base extension (SBE), allele-specific primer extension (ASPE), oligonucleotide ligation (OL), and direct hybridization (DH). All four assay methods used the same Luminex 100 flow cytometer platform. Fifty-eight F₂-derived soybean [Glycine max (L.) Merr.] lines from a cross between inbred lines G99-G725 and N00-3350 were genotyped at four SNPs. SBE and ASPE clearly differentiated between the two homozygotes and the heterozygote at each SNP. Results were in agreement with those identified using the SNaPshot minisequencing assay as a control. In contrast, the OL and DH assays were unable to differentiate between genotypes at some of the SNPs. However, when the cost per data point for the four different assays was compared, the cost of OL and DH was only about 70% of that for SBE, with DH requiring the least time of the four assays. On the basis of cost and labor, ASPE is more cost-effective and simpler than SBE, and would therefore be a good method for genetic mapping and diversity studies which require a large number of markers and a high level of multiplexing. DH appears to be the most economical assay for marker-assisted selection, though optimization for DH would be required for some SNP markers.     

Development and assessment of simple PCR markers for SNP genotyping in barley

Simple molecular marker assays underpin routine plant breeding and research activities in many laboratories worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important crop plants, the availability of routine, low-tech marker assays for genotyping SNPs is of increased importance. In this study, we demonstrate that temperature-switch PCR (TSP) supports the rapid development of robust, allele-specific PCR markers for codominant SNP genotyping on agarose gel. A total of 87 TSP markers for assessing gene diversity in barley were developed and used to investigate the efficacy for marker development, assay reliably and genotyping accuracy. The TSP markers described provide good coverage of the barley genome, are simple to use, easy to interpret and score, and are amenable to assay automation. They provide a resource of informative SNP markers for assessing genetic relationships among individuals, populations and gene pools of cultivated barley (Hordeum vulgare L.) and its wild relative H. spontaneum K. Koch. TSP markers provide opportunities to use available SNP resources for marker-assisted breeding and plant genetic research, and to generate information that can be integrated with SNP data from different sources and studies. TSP markers are expected to provide similar advantages for any animal or plant species. M. J. Hayden and T. Tabone contributed equally to this work.     

Evaluation of linkage disequilibrium and its effect on non-parametric multipoint linkage analysis using two high density single-nucleotide polymorphism mapping panels

Genotype data from the Illumina Linkage III SNP panel (n = 4,720 SNPs) and the Affymetrix 10 k mapping array (n = 11,120 SNPs) were used to test the effects of linkage disequilibrium (LD) between SNPs in a linkage analysis in the Collaborative Study on the Genetics of Alcoholism pedigree collection (143 pedigrees; 1,614 individuals). The average r2 between adjacent markers across the genetic map was 0.099 +/- 0.003 in the Illumina III panel and 0.17 +/- 0.003 in the Affymetrix 10 k array. In order to determine the effect of LD between marker loci in a nonparametric multipoint linkage analysis, markers in strong LD with another marker (r2 > 0.40) were removed (n = 471 loci in the Illumina panel; n = 1,804 loci in the Affymetrix panel) and the linkage analysis results were compared to the results using the entire marker sets. In all analyses using the ALDX1 phenotype, 8 linkage regions on 5 chromosomes (2, 7, 10, 11, X) were detected (peak markers p < 0.01), and the Illumina panel detected an additional region on chromosome 6. Analysis of the same pedigree set and ALDX1 phenotype using short tandem repeat markers (STRs) resulted in 3 linkage regions on 3 chromosomes (peak markers p < 0.01). These results suggest that in this pedigree set, LD between loci with spacing similar to the SNP panels tested may not significantly affect the overall detection of linkage regions in a genome scan. Moreover, since the data quality and information content are greatly improved in the SNP panels over STR genotyping methods, new linkage regions may be identified due to higher information content and data quality in a dense SNP linkage panel.     

SNP marker detection and genotyping in tilapia

We have generated a unique resource consisting of nearly 175 000 short contig sequences and 3569 SNP markers from the widely cultured GIFT (Genetically Improved Farmed Tilapia) strain of Nile tilapia (Oreochromis niloticus). In total, 384 SNPs were selected to monitor the wider applicability of the SNPs by genotyping tilapia individuals from different strains and different geographical locations. In all strains and species tested (O. niloticus, O. aureus and O. mossambicus), the genotyping assay was working for a similar number of SNPs (288–305 SNPs). The actual number of polymorphic SNPs was, as expected, highest for individuals from the GIFT population (255 SNPs). In the individuals from an Egyptian strain and in individuals caught in the wild in the basin of the river Volta, 197 and 163 SNPs were polymorphic, respectively. A pairwise calculation of Nei’s genetic distance allowed the discrimination of the individual strains and species based on the genotypes determined with the SNP set. We expect that this set will be widely applicable for use in tilapia aquaculture, e.g. for pedigree reconstruction. In addition, this set is currently used for assaying the genetic diversity of native Nile tilapia in areas where tilapia is, or will be, introduced in aquaculture projects. This allows the tracing of escapees from aquaculture and the monitoring of effects of introgression and hybridization.