Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome‐wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red‐tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site‐associated DNA marker approach (DArTseq^?). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq‐based method to produce reliable genome‐wide SNP data from traditional museum specimens.     

Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses

Noninvasive faecal DNA sampling has the potential to provide a wealth of information necessary for monitoring and managing endangered species while eliminating the need to capture, handle or observe rare individuals. However, scoring problems, and subsequent genotyping errors, associated with this monitoring method remain a great concern as they can lead to misidentification of individuals and biased estimates. We examined a kit fox scat data set (353 scats; 80 genotypes) for genotyping errors using both genetic and GIS analyses, and evaluated the feasibility of combining both approaches to assess reliability of the faecal DNA results. We further checked the appropriateness of using faecal genotypes to study kit fox populations by describing information about foxes that we could deduce from the 'acceptable' scat genotypes, and comparing it to information gathered with traditional field techniques. Overall, genetic tests indicated that our data set had a low rate of genotyping error. Furthermore, examination of distributions of scat locations confirmed our data set was relatively error free. We found that analysing information on sex primer consistency and scat locations provided a useful assessment of scat genotype error, and greatly limited the amount of additional laboratory work that was needed to identify potentially 'false' scores. 'Acceptable' scat genotypes revealed information on sex ratio, relatedness, fox movement patterns, latrine use, and size of home range. Results from genetic and field data were consistent, supporting the conclusion that our data set had a very low rate of genotyping error and that this noninvasive method is a reliable approach for monitoring kit foxes.     

Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet

The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long‐term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage.     

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.     

LSGermOPA,a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) germplasm fingerprinting

To deploy a high-throughput genotyping platform in germplasm management, we designed and tested a custom OPA (Oligo Pool All), LSGermOPA, for assessing the genetic diversity and population structure of the USDA cultivated lettuce (Lactuca sativa L.) germplasm collection using Illumina’s GoldenGate assay. This OPA contains 384 EST (expressed sequence tag)-derived SNP (single nucleotide polymorphism) markers selected from a large set of SNP markers experimentally validated and mapped by the Compositae Genome Project. Used for genotyping were DNA samples prepared from bulked leaves of five randomly-selected seedlings from each of 380 lettuce accessions. High-quality genotype data were obtained from 354 of the 384 SNPs. The reproducibility of automatic genotype calls was 99.8% as calculated from the four pairs of duplicated DNA samples in the assay. An unexpectedly high percentage of heterozygous genotypes at the polymorphic loci for most accessions indicated a high level of heterogeneity within accessions. Only 148 homogenous accessions, collectively comprising all five horticultural types, were used in subsequent analyses to demonstrate the usefulness of LSGermOPA. The results of phylogenetic relationship, population structure and genetic differentiation analyses were consistent with previous reports using other marker systems. This suggests that LSGermOPA is capable of revealing sufficient levels of polymorphism among lettuce cultivars and is appropriate for rapid assessment of genetic diversity and population structure in the lettuce germplasm collection. Challenges and strategies for effective genotyping and managing lettuce germplasm are discussed.     

Amplification success of multilocus genotypes from feathers found in the field compared with feathers obtained from shot birds

Effective DNA extraction methods from bird feathers have facilitated non‐invasive sampling, leading to the suggestion that feathers are a great source for genetic studies. However, few studies have assessed whether all feathers can be used or provide equal numbers of useful templates. In this study, feathers collected in various ways from Red Grouse Lagopus lagopus were examined to establish the quality of DNA extracted. Individual samples were classified into two categories according to whether they were collected from shot birds or found in the field. DNA was extracted from all samples and genotyped at 19 microsatellite loci. PCR products were analysed on a MegaBACE 1000. A total of 93% of the ‘shot’ category produced a genotype that was considered successful (i.e. 15 of 18 loci) and 23% of the ‘collected’ category produced successful genotypes under the same criteria. There was a significant difference between shot and collected samples in genotyping success and the observed number of missing loci. Recommendations and best practices are discussed along with the utility of bird feathers as a source of DNA for population and conservation biology.     

Developing genomic resources for the common bottlenose dolphin (Tursiops truncatus): isolation and characterization of 153 single nucleotide polymorphisms and 53 genotyping assays

Although single nucleotide polymorphisms (SNPs) are commonly used in human genetics, they have only recently been incorporated into genetic studies of non‐model organisms, including cetaceans. SNPs have several advantages over other molecular markers for studies of population genetics: they are quicker and more straightforward to score, cross‐laboratory comparisons of data are less complicated, and they can be used successfully with low‐quality DNA. We screened portions of the genome of one of the most abundant cetaceans in U.S. waters, the common bottlenose dolphin (Tursiops truncatus), and identified 153 SNPs resulting in an overall average of one SNP every 463 base pairs. Custom TaqMan^® Assays were designed for 53 of these SNPs, and their performance was tested by genotyping a set of bottlenose dolphin samples, including some with low‐quality DNA. We found that in 19% of the loci examined, the minor allele frequency (MAF) estimated during initial SNP ascertainment using a DNA pool of 10 individuals differed significantly from the final MAF after genotyping over 100 individuals, suggesting caution when making inferences about MAF values based on small data sets. For two assays, we also characterized the basis for unusual clustering patterns to determine whether their data could still be utilized for further genetic studies. Overall results support the use of these SNPs for accurate analysis of both poor and good‐quality DNA. We report the first SNP markers and genotyping assays for use in population and conservation genetic studies of bottlenose dolphins.     

Genome-wide SNP loci reveal novel insights into koala (<Emphasis Type="Italic">Phascolarctos cinereus</Emphasis>) population variability across its range

The koala (Phascolarctos cinereus) is an iconic Australian species that is currently undergoing a number of threatening processes, including disease and habitat loss. A thorough understanding of population genetic structuring and genomic variability of this species is essential to effectively manage populations across the species range. Using a reduced representation genome sequencing method known as double digest restriction-associated sequencing, this study has provided the first genome-wide SNP marker panel in the koala. In this study, 33,019 loci were identified in the koala and a filtered panel of 3060 high-utility SNP markers, including 95 sex-linked markers, were used to provide key insights into population variability and genomic variation in 171 koalas from eight populations across their geographic range. Broad-scale genetic differentiation between geographically separated populations (including sub-species) was assessed and revealed significant differentiation between all populations (F_ST range = 0.01–0.28), with the largest divergence observed between the three geographically distant subgroups (QLD, NSW and VIC) along the east coast of Australia (average F_ST range = 0.17–0.23). Sub-group divergence appears to be a reflection of an isolation by distance effect and sampling strategy rather than true evidence of sub-speciation. This is further supported by low proportions of AMOVA variation between sub-species groups (11.19 %). Fine-scale analysis using genome-wide SNP loci and the NETVIEW pipeline revealed cryptic genetic sub-structuring within localised geographic regions, which corresponded to the hierarchical mating system of the species. High levels of genome-wide SNP heterozygosity were observed amongst all populations (He = 0.25–0.35), and when evaluating across the species to other vertebrate taxa were amongst the highest values observed. This illustrates that the species as a whole still retains high levels of diversity which is comparable to other outbred vertebrate taxa for genome-wide SNPs. Insights into the potential for adaptive variation in the koala were also gained using outlier analysis of genome-wide SNPs. A total of 10 putative outlier SNPs were identified indicating the high likelihood of local adaptations within populations and regions. This is the first use of genome-wide markers to assess population differentiation at a broad-scale in the koala and the first time that sex-linked SNPs have been identified in this species. The application of this novel genomic resource to populations across the species range will provide in-depth information allowing informed conservation priorities and management plans for in situ koalas across Australia and ex situ around the world.     

SNP discovery in wild and domesticated populations of blue catfish,Ictalurus furcatus,using genotyping‐by‐sequencing and subsequent SNP validation

Blue catfish, Ictalurus furcatus, are valued in the United States as a trophy fishery for their capacity to reach large sizes, sometimes exceeding 45 kg. Additionally, blue catfish × channel catfish (I. punctatus) hybrid food fish production has recently increased the demand for blue catfish broodstock. However, there has been little study of the genetic impacts and interaction of farmed, introduced and stocked populations of blue catfish. We utilized genotyping‐by‐sequencing (GBS) to capture and genotype SNP markers on 190 individuals from five wild and domesticated populations (Mississippi River, Missouri, D&B, Rio Grande and Texas). Stringent filtering of SNP‐calling parameters resulted in 4275 SNP loci represented across all five populations. Population genetics and structure analyses revealed potential shared ancestry and admixture between populations. We utilized the Sequenom MassARRAY to validate two multiplex panels of SNPs selected from the GBS data. Selection criteria included SNPs shared between populations, SNPs specific to populations, number of reads per individual and number of individuals genotyped by GBS. Putative SNPs were validated in the discovery population and in two additional populations not used in the GBS analysis. A total of 64 SNPs were genotyped successfully in 191 individuals from nine populations. Our results should guide the development of highly informative, flexible genotyping multiplexes for blue catfish from the larger GBS SNP set as well as provide an example of a rapid, low‐cost approach to generate and genotype informative marker loci in aquatic species with minimal previous genetic information.     

Genetic structure and diversity of the koala population in South Gippsland,Victoria: a remnant population of high conservation significance

In the Australian state of Victoria, the history of koalas and their management has resulted in the homogenisation and reduction of genetic diversity in many contemporary populations. Decreased genetic diversity may reduce a species’ ability to adapt to future environmental pressures such as climate change or disease. The South Gippsland koala population is considered to be unique in Victoria, as it is believed to be a remnant population, not originating from managed populations that have low genetic variation. This study investigated genetic structure and diversity of koalas in South Gippsland, with comparison to other populations in Victoria (French Island/Cape Otway, FI and Raymond Island, RI), New South Wales and south east Queensland. Population analyses were undertaken using both microsatellite genotype and mitochondrial DNA sequence data. Non-invasive sampling of koala scats was used to source koala DNA, allowing 222 South Gippsland koalas to be genotyped. Using nuclear data the South Gippsland koala population was found to be significantly differentiated (D_jost 95% CI SG–RI?=?0.03–0.06 and SG–FI?=?0.08–012) and more diverse (A_R 95% CI SG?=?4.7–5.6, RI?=?3.1–3.3, FI?=?3.0–3.3; p?=?0.001) than other Victorian koala populations, supporting the premise that koalas in the South Gippsland region are part of a remnant population, not derived from translocated island stock. These results were also supported by mitochondrial data where eight haplotypes (Pc4, Pc17, Pc26, Pc27, and Pc56–Pc59) were identified in South Gippsland while a single haplotype (Pc27) was found in all island koalas tested. Compared to other Victorian koala populations, greater genetic diversity found in South Gippsland koalas, may provide this population with a greater chance of survival in the face of future environmental pressures. The South Gippsland koala population is, therefore, of high conservation significance, warranting the implementation of strategies to conserve this population and its diversity into the future.     

三江源和祁连山国家公园雪豹种群的遗传结构分析

掌握遗传信息对濒危物种的保护和管理具有重要意义。本研究在我国雪豹重要分布区祁连山和三江源国家公园分别采集粪便样品,利用mtDNA的cyt b基因、微卫星多态性位点进行了雪豹的物种鉴定、个体识别和种群遗传结构评估。在采集286份疑似雪豹粪便样品中,成功的对86份雪豹样品进行了扩增鉴定,利用微卫星位点进行个体识别获得41只雪豹个体,其中祁连山国家公园26只,三江源国家公园15只。通过等位基因数、有效等位基因数、观测杂合度、期望杂合度、多态信息含量等指标进行种群遗传多样性评估,认为雪豹种群遗传多样性相对较低,但祁连山国家公园雪豹种群遗传多样性相对较高。STRUCTURE进行群体遗传结构分析表明,4个种群可以划分为3个遗传类群,祁连山国家公园的种群（YCW和QLS）与三江源国家种群(DC和SJ)的遗传差异,可能与种群间的地理隔离存在明显的相似性。     

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。     

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Background

Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen^® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR^® Identifiler^® panel) and N = 49 SNP (TaqMan^®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.

Results

The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.

Conclusion

The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan^® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR^® Identifiler^® panel from wgaDNA equivalent to that of gDNA.

     

Genotyping‐in‐Thousands by sequencing (GT‐seq) panel development and application to minimally invasive DNA samples to support studies in molecular ecology

Minimally invasive sampling (MIS) is widespread in wildlife studies; however, its utility for massively parallel DNA sequencing (MPS) is limited. Poor sample quality and contamination by exogenous DNA can make MIS challenging to use with modern genotyping‐by‐sequencing approaches, which have been traditionally developed for high‐quality DNA sources. Given that MIS is often more appropriate in many contexts, there is a need to make such samples practical for harnessing MPS. Here, we test the ability for Genotyping‐in‐Thousands by sequencing (GT‐seq), a multiplex amplicon sequencing approach, to effectively genotype minimally invasive cloacal DNA samples collected from the Western Rattlesnake (Crotalus oreganus), a threatened species in British Columbia, Canada. As there was no previous genetic information for this species, an optimized panel of 362 SNPs was selected for use with GT‐seq from a de novo restriction site‐associated DNA sequencing (RADseq) assembly. Comparisons of genotypes generated within and among RADseq and GT‐seq for the same individuals found low rates of genotyping error (GT‐seq: 0.50%; RADseq: 0.80%) and discordance (2.57%), the latter likely due to the different genotype calling models employed. GT‐seq mean genotype discordance between blood and cloacal swab samples collected from the same individuals was also minimal (1.37%). Estimates of population diversity parameters were similar across GT‐seq and RADseq data sets, as were inferred patterns of population structure. Overall, GT‐seq can be effectively applied to low‐quality DNA samples, minimizing the inefficiencies presented by exogenous DNA typically found in minimally invasive samples and continuing the expansion of molecular ecology and conservation genetics in the genomics era.     

Is genomic diversity a useful proxy for census population size? Evidence from a species‐rich community of desert lizards

Species abundance data are critical for testing ecological theory, but obtaining accurate empirical estimates for many taxa is challenging. Proxies for species abundance can help researchers circumvent time and cost constraints that are prohibitive for long‐term sampling. Under simple demographic models, genetic diversity is expected to correlate with census size, such that genome‐wide heterozygosity may provide a surrogate measure of species abundance. We tested whether nucleotide diversity is correlated with long‐term estimates of abundance, occupancy and degree of ecological specialization in a diverse lizard community from arid Australia. Using targeted sequence capture, we obtained estimates of genomic diversity from 30 species of lizards, recovering an average of 5,066 loci covering 3.6 Mb of DNA sequence per individual. We compared measures of individual heterozygosity to a metric of habitat specialization to investigate whether ecological preference exerts a measurable effect on genetic diversity. We find that heterozygosity is significantly correlated with species abundance and occupancy, but not habitat specialization. Demonstrating the power of genomic sampling, the correlation between heterozygosity and abundance/occupancy emerged from considering just one or two individuals per species. However, genetic diversity does no better at predicting abundance than a single day of traditional sampling in this community. We conclude that genetic diversity is a useful proxy for regional‐scale species abundance and occupancy, but a large amount of unexplained variation in heterozygosity suggests additional constraints or a failure of ecological sampling to adequately capture variation in true population size.     

Non-toxic and efficient DNA extractions for soybean leaf and seed chips for high-throughput and large-scale genotyping

In applied soybean (Glycine max L.) breeding programs, marker-assisted selection has become a necessity to select value-added quantitative trait loci. The goal of this work was to improve marker-assisted selection workflow by developing a reliable, inexpensive, high-throughput DNA extraction protocol for soybean seed and leaf samples that does not generate hazardous waste. The DNA extraction protocol developed allows for the leverage of robust SNP genotyping platforms such as the Simple Probe Assay and KASPar v4.0 SNP Genotyping System to genotype thousands of seeds or leaves non-destructively in a single day with a 95 % success rate. This methodology makes it possible to run up to 150 SNP markers on the DNA extracted from a single seed chip or leaf sample.     

Characterization of polyploid wheat genomic diversity using a high‐density 90 000 single nucleotide polymorphism array

High‐density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker–trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene‐associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome‐wide distributed SNPs that are represented in populations of diverse geographical origin. We used density‐based spatial clustering algorithms to enable high‐throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model‐free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low‐intensity clusters can provide insight into the distribution of presence–absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.     

高保真酶特异性检测大鼠SNP基因型新方法

目的应用高保真酶（Pfu）和3’末端修饰引物在单管双向等位基因特异性扩增（SB-ASA）中区分SNP基因型,建立高保真酶特异性检测SNP基因型的新方法。方法选取近交系大鼠SNP位点,以RS8149053为例,设计两个外部引物和两个等位基因特异性引物,四引物3’末端进行硫代磷酸化修饰,应用高保真聚合酶（Pfu）进行特异性扩增,扩增结果测序验证其可靠性。结果在RS8149053 SNP位点（C/T）上,等位基因型CC扩增出179 bp目的片段,基因型TT扩增出597 bp目的片段,基因型不同则扩增出分子量不同的片段,目的条带测序结果与Rat Genome Database数据库基因型结果一致,高保真酶扩增结果稳定且特异性强。结论高保真酶等位基因特异性扩增技术能有效降低假阳性率,是一种快速、特异的SNP基因分型新方法。     

Gene mapping in the wild with SNPs: guidelines and future directions

One of the biggest challenges facing evolutionary biologists is to identify and understand loci that explain fitness variation in natural populations. This review describes how genetic (linkage) mapping with single nucleotide polymorphism (SNP) markers can lead to great progress in this area. Strategies for SNP discovery and SNP genotyping are described and an overview of how to model SNP genotype information in mapping studies is presented. Finally, the opportunity afforded by new generation sequencing and typing technologies to map fitness genes by genome-wide association studies is discussed.     

Highly accurate SNP genotyping from historical and low‐quality samples

Historical and other poor‐quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error‐prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor‐quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual‐fluorescence genotyping that is very effective for genotyping poor‐quality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/µL of a 51‐bp nuclear sequence. Among samples with ≤ 10 copies/µL DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.